Broad-spectrum responsive self-assembling peptide and application thereof

Abstract

The invention relates to a broad-spectrum responsive self-assembling peptide, a hydrogel formed by the self-assembling peptide, and an application of the hydrogel.

Description

Broad-spectrum responsive self-assembling peptides and their applications Technical Field

[0001] The present invention relates to the field of biomedical materials, in particular to broad-spectrum responsive peptide hydrogel biomaterials. Background Art

[0002] As a three-dimensional scaffold material, self-assembling peptide hydrogels have excellent biocompatibility and functional diversity and are widely used in biomedical fields such as 3D cell culture, drug delivery, drug screening and evaluation, and tissue engineering. Among them, stimulus-responsive peptides have attracted much attention due to their controllable gelation process. However, the gelation of self-assembling peptides often depends on temperature (CN 202010572759.2), pH changes (such as the peptide hydrogel described in US 201 / 03262451 A1 changes with pH changes, and the peptide hydrogel described in CN201680012178.5 needs to be used when pH < 3.5), increasing or decreasing specific light (the gelation of the peptide in CN201680061179.9 depends on photoactivation), the introduction of metal salt ions (US 2011 / 0165200 A1 proposes a series of peptides including MAX1, which can coordinate with calcium ions for gelation; the peptide hydrogel for skin repair in CN 202110863352.X requires a specific sodium ion concentration) or some exogenous substances (CN201810783581.9 involves a peptide composite hydrogel, and the gelation process requires the introduction of calcium phosphate), etc. Such strict gelation conditions are accompanied by more complicated usage processes and bring additional risks. When using these hydrogels in biological experiments, non-physiological temperatures and pH levels pose a significant threat to cell activity; photoresponsive gelation relies on complex and expensive peripheral devices; high metal salt ion concentrations affect the osmotic pressure of the cellular microenvironment; and exogenous substances may induce immune responses, posing certain safety risks. Existing peptides capable of gelation in response to endogenous substances are relatively few in number, and most are limited to a single endogenous substance, limiting their scope of application.

[0003] In summary, the application of stimuli-responsive polypeptide hydrogels is limited by complex response initiation that is not suitable for application under physiological conditions, or by the endogenous single gelation conditions.

[0004] Summary of the Invention

[0005] The present invention provides a broad-spectrum responsive self-assembling peptide, which can be triggered by a broad spectrum of triggering substances and respond to self-assembly to form a nano-network structure, and a self-assembling peptide solution system that can exert supporting and repair functions in a solution state.

[0006] In a first aspect, the present invention provides a broad-spectrum responsive self-assembling peptide, wherein the self-assembling peptide comprises a hydrophobic domain and a hydrophilic domain, wherein the hydrophilic domain comprises at least two consecutive β-turn regions capable of forming β-turns.

[0007] In some embodiments, the at least one β-turn region comprises or is linked to one or more acidic amino acids at a terminal end, preferably comprises or is linked to one acidic amino acid.

[0008] In some embodiments, at least one β-turn region comprises an acidic amino acid at a terminal end.

[0009] In some embodiments, the β-turn region comprises a β-turn motif formed by 3-6 amino acids, and the β-turn motif has the following structure:

[0010] X1X2X3, X1X2X3X4, X1X2X3X4X5, or X1X2X3X4X5X6,

[0011] Wherein, X1, X2, X3, X4, X5, and X6 are amino acid residues, and X1, X2, X3, X4, X5, and X6 in each β-turn motif are identical to or different from each other.

[0012] In some embodiments, the β-turn motif comprises one hydroxyproline (O), and preferably, X2 is hydroxyproline (O).

[0013] In some embodiments, the hydrophilic domain comprises 2-8 β-turn regions.

[0014] In some embodiments, the hydrophilic domain comprises 2, 3, 4, 5, 6, 7, or 8 β-turn regions.

[0015] Preferably, the hydrophilic domain comprises 2-6 β-turn regions. More preferably, the hydrophilic domain comprises 2-4 β-turn regions.

[0016] Preferably, the hydrophilic domain comprises 2 or 3 β-turn regions.

[0017] In some embodiments, the β-turn motif in the at least one β-turn region comprises or is linked to an acidic amino acid.

[0018] In some embodiments, the β-turn motif in the at least one β-turn region comprises or is linked to a glutamic acid (E), valine (V), leucine (L), isoleucine (I), aspartic acid (D) or lysine (K).

[0019] In some embodiments, the hydrophilic domain comprises two β-turn regions, and the ends of the β-turn regions comprise acidic amino acids E.

[0020] In some embodiments, the hydrophilic domain comprises two β-turn regions, wherein one end of the β-turn region comprises an acidic amino acid E, and the other end of the β-turn region comprises an amino acid V or K.

[0021] In some embodiments, the hydrophilic domain comprises two β-turn regions, and the ends of the β-turn regions comprise acidic amino acids D.

[0022] In some embodiments, the hydrophilic domain comprises two β-turn regions, wherein one β-turn region comprises an acidic amino acid D at its end, and the other β-turn region comprises an amino acid V at its end.

[0023] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X2 is hydroxyproline O, or the hydrophilic domain comprises at least one β-turn motif in which X2 is proline P.

[0024] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X2 is O.

[0025] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X1 is G.

[0026] In some embodiments, the hydrophilic domain comprises a β-turn motif in which X2 is O and a β-turn motif in which X2 is P.

[0027] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X2 is P.

[0028] In some embodiments, the hydrophilic domain comprises two β-turn motifs in which X2 is P.

[0029] In some embodiments, one or more of X1, X3 and X4 is glycine (G), and / or one or both of X3 and X4 is alanine (A).

[0030] In some embodiments, the β-turn motif comprises an amino acid sequence selected from the group consisting of:

[0031] GPGG (SEQ ID NO.: 33), GPGA (SEQ ID NO.: 34), GPAG (SEQ ID NO.: 35), GPG, GPAA (SEQ ID NO.: 36), GPGGG (SEQ ID NO.: 37), GOGG (SEQ ID NO.: 38), GOGA (SEQ ID NO.: 39), GOAG (SEQ ID NO.: 40), GOGGA (SEQ ID NO.: 37) NO.: 41), GOAA (SEQ ID NO.: 42), GOG, or GOGV (SEQ ID NO.: 43);

[0032] Preferably, the β-turn motif has an amino acid sequence selected from the group consisting of:

[0033] GPAGE (SEQ ID NO.: 44), GPGGE (SEQ ID NO.: 45), GOGAE (SEQ ID NO.: 46), GOGGAE (SEQ ID NO.: 47), GOGE (SEQ ID NO.: 48), GOGGE (SEQ ID NO.: 49), GPGAD (SEQ ID NO.: 50), GOGGD (SEQ ID NO.: 51), GPGGV (SEQ ID NO.: 51) NO.:52), GOGGV (SEQ ID NO.:53), GPGGK (SEQ ID NO.:54), GOGGK (SEQ ID NO.:55), GPGAE (SEQ ID NO.:56), GOGAD (SEQ ID NO.:57), GPAAD (SEQ ID NO.:58), GOAAE (SEQ ID NO.:59), GPGGD (SEQ ID NO.: 60), GPGGGV (SEQ ID NO.: 61), GPGV (SEQ ID NO.: 62), GOGGI (SEQ ID NO.: 63) or GOGVI (SEQ ID NO.: 64).

[0034] In some embodiments, X1 and X4 form a hydrogen bond.

[0035] In some embodiments, the hydrophilic domain comprises 2, 3, 4, 5, 6, 7 or 8 β-turn motifs, preferably, the hydrophilic domain comprises 2 or 3 β-turn motifs.

[0036] In some embodiments, the beta-turn motif has an amino acid sequence selected from the group consisting of:

[0037] GOGG (SEQ ID NO.:38), GPGG (SEQ ID NO.:33), GOGA (SEQ ID NO.:39), GOAG (SEQ ID NO.:40), GPGA (SEQ ID NO.:34) or GPAG (SEQ ID NO.:35).

[0038] At least one beta-turn motif in the hydrophilic domain includes an alanine, thereby improving the mechanical properties of the self-assembling peptide.

[0039] In some embodiments, the C-terminus of the hydrophilic domain can be modified with an agent or group selected from the group consisting of carboxylic acid, thiol, ketoate, nitrite, phosphonate, thiophosphate, carbonate, sulfate, nitrate, vinyl sulfone, amide, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and / or silane modifications.

[0040] The amino acid sequence of the hydrophilic domain is more hydrophilic than the amino acid sequence of the hydrophobic domain.

[0041] In some embodiments, the hydrophobic domain comprises 3-10 hydrophobic amino acids. Preferably, the hydrophobic domain comprises 3-7 hydrophobic amino acids. Preferably, the hydrophobic domain comprises 3-5 hydrophobic amino acids. More preferably, the hydrophobic domain comprises 5 hydrophobic amino acids.

[0042] Preferably, the hydrophobic amino acid is selected from one or more of isoleucine (I), valine (V), leucine (L), phenylalanine (F) and alanine (A).

[0043] In some embodiments, the hydrophobic amino acid is selected from one or more of I, V, L, A, and F.

[0044] In some embodiments, the N-terminus of the hydrophobic domain is modified with an agent or group selected from the group consisting of acetyl, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and / or silane modifications.

[0045] In some embodiments, the hydrophobic domain has an amino acid sequence selected from the group consisting of:

[0046] LLLL (SEQ ID NO.: 65), FIIII (SEQ ID NO.: 66), IIII (SEQ ID NO.: 67), IIII (SEQ ID NO.: 68), ILILI (SEQ ID NO.: 69), FLFLF (SEQ ID NO.: 70), IVIVI (SEQ ID NO.: 71), VIVIV (SEQ ID NO.: 72), VLFIIV (SEQ ID NO.: 72), NO.:73), VLIII (SEQ ID NO.:74), IVALF (SEQ ID NO.:75), LFIVL (SEQ ID NO.:76), FIAIV (SEQ ID NO.:77), FIIIV (SEQ ID NO.:78), Ac-VLFIIV (SEQ ID NO.:79), Ac-IVIVI (SEQ ID NO.:80), Ac-IIIIII (SEQ ID NO.:78) NO.: 81), IIIIII (SEQ ID NO.: 82), FLIVI (SEQ ID NO.:83), FLIIA (SEQ ID NO.:84), FIFIF (SEQ ID NO.:85), IFIFI (SEQ ID NO.:86), IAILI (SEQ ID NO.:87) or LLLLL (SEQ ID NO.:88).

[0047] The amino acid sequence of the hydrophobic domain is hydrophobic compared to the amino acid sequence of the hydrophilic domain.

[0048] In some embodiments, the self-assembling peptide further comprises a linker domain providing a spacer between the hydrophobic domain and the hydrophilic domain.

[0049] In some embodiments, the linker domain comprises 2-8 amino acid residues, preferably 4-5 amino acid residues.

[0050] In some embodiments, the linker domain comprises amino acids with small side chains, amino acids with hydroxyl groups on their side chains, and / or hydrophobic amino acids that are distal to the hydrophobic region.

[0051] In some embodiments, the amino acid with a smaller side chain is selected from glycine (G), alanine (A), and serine (S).

[0052] In some embodiments, the amino acid with a hydroxyl group on the side chain is selected from serine (S), threonine (T) and hydroxyproline (O),

[0053] In some embodiments, the hydrophobic amino acids away from the hydrophobic domain are selected from I, V, L, F and A, and the hydrophobic amino acids I, V, F, L, and A are interchangeable.

[0054] In some embodiments, the connecting domain has an amino acid sequence selected from the group consisting of:

[0055] GSII (SEQ ID NO.: 89), GPOGI (SEQ ID NO.: 90, GPOGV (SEQ ID NO.: 91), GSGII (SEQ ID NO.: 92), GSVI (SEQ ID NO.: 93), GOII (SEQ ID NO.: 94), GPOGL (SEQ ID NO.: 95), OGII (SEQ ID NO.: 96) or GTVI (SEQ ID NO.: 97), wherein S, T, and O are interchangeable;

[0056] More preferably, the connecting domain has an amino acid sequence selected from the group consisting of:

[0057] GSII (SEQ ID NO.: 89), GTII (SEQ ID NO.: 98), GTVI (SEQ ID NO.: 97), GOVI (SEQ ID NO.: 99), GSVI (SEQ ID NO.: 93), GSVL (SEQ ID NO.: 100), GSGII (SEQ ID NO.: 92), GSGVI (SEQ ID NO.: 101), GOII (SEQ ID NO.: 101) NO.: 94), OGII (SEQ ID NO.: 96), GOGVI (SEQ ID NO.: 102) or GOGII (SEQ ID NO.: 103).

[0058] In some embodiments, one or more Gs are further included between the hydrophobic domain and the connecting domain to enhance the softness and flexibility of the self-assembling peptide.

[0059] In some embodiments, the self-assembling peptide has a length of 15-50 amino acids, preferably 15-25 amino acids.

[0060] In some embodiments, the self-assembling peptide comprises 2, 3, 4, 5, 6, 7 or 8 β-turns. Preferably, the self-assembling peptide comprises 2 or 3 β-turns.

[0061] In some embodiments, the self-assembling peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32:

[0062] IIIIIIGSIIGPGGDGPGGV (SEQ ID NO.1),

[0063] IIIIIGSIIGPGGEGPGGV(SEQ ID NO.2)、

[0064] IIIIGSIIGOGGEGPGGV(SEQ ID NO.3)、

[0065] IIIIGSIIGOGGEGPGGV(SEQ ID NO.4)、

[0066] IIIIGSIIGOGGEGPGGGV(SEQ ID NO.5)、

[0067] IIIIIGSIIGOGGEGPGV(SEQ ID NO.6)、

[0068] IIIIIGIIGOGAEGPGGV(SEQ ID NO.7)、

[0069] IIIIIGSIIOGGAEGPGGV(SEQ ID NO.8)、

[0070] IIIIGSIIGOGGVGPGGV(SEQ ID NO.9)、

[0071] IIIIIIGSIIGOGAEGPGGVGPGGV(SEQ ID NO.10)

[0072] FLIGHTING EGPGGV(SEQ ID NO.11)、

[0073] FLIIAGSIIGPGGDGOGGV(SEQ ID NO.12)

[0074] IIIIIGOGIIGPGGEGPGGE(SEQ ID NO.13)、

[0075] IIIIIGTVIGPGGEGOGGE(SEQ ID NO.14)、

[0076] IIIIIGSIIGOGGEGPGGE(SEQ ID NO.15)、

[0077] IAILIGTVIGPGGEGOGGE(SEQ ID NO.16)、

[0078] IIIIGTVIGPGGEGOGGV(SEQ ID NO.17)、

[0079] IVIVIGSIIGOGGDGPGGV(SEQ ID NO.18)、

[0080] IVIVIGSIIGPGGEGOGGV (SEQ ID NO.19),

[0081] FLIVIGOGIIGOGGEGPGGE(SEQ ID NO.20)

[0082] IVIVIGSGIIGPGGDGPGGV (SEQ ID NO. 21),

[0083] IVIVIGIGIIGOGGDGOGGV (SEQ ID NO.22),

[0084] IIIIIGSIIGPGGEGOGGV (SEQ ID NO. 23),

[0085] FIIIVGSIIGPGGEGPGGE(SEQ ID NO.24),

[0086] IIIIIIGOGIIGOGGEGPGGV (SEQ ID NO.25),

[0087] Ac-IIIIGSIIGPGGEGOGGV (SEQ ID NO. 26),

[0088] FLIVIGSIIGOGAEGPGGV (SEQ ID NO.27),

[0089] IVIVIGSGIIGPGGEGPGGV (SEQ ID NO.28),

[0090] LLLLLSVLGPAGEGPAGE (SEQ ID NO.: 29),

[0091] LLLLLGPOGLGPAGEGPAGE (SEQ ID NO.: 30),

[0092] LLLLLGPOGVGPAGEGPAGE (SEQ ID NO.: 31) or

[0093] LLLLLGPOGIGPAGEGPAGE (SEQ ID NO.: 32).

[0094] The self-assembling peptide of the present invention having the above structure can be initiated by a broad-spectrum initiator to form a three-dimensional network scaffold material.

[0095] The three-dimensional mesh scaffold material has a nanostructure.

[0096] In some embodiments, the broad-spectrum initiator is a positive charge source substance or a mixed system comprising a positive charge source substance, wherein the positive charge source substance includes a substance with a positively charged group or a positively charged ion.

[0097] In some embodiments, the positive charge source material is a biomacromolecule, drug, functional molecule, animal tissue fluid, or a cell culture storage or drug and functional molecule delivery system containing the above components, whose number of hydrogen bond acceptors is less than the number of hydrogen bond donors under neutral conditions.

[0098] In some embodiments, the broad-spectrum triggering substance is selected from physiological molecules, biochemical molecules, drugs, functional molecules, metal ions, microparticles or microspheres with positively charged surfaces containing positive electrochemical groups, or a mixed system of one or more thereof, or a mixed system of one or more of animal tissue fluid, complete cell culture medium, serum-free culture medium, cell culture storage fluid, or drug and functional molecule delivery systems containing the aforementioned substances.

[0099] In some embodiments, the broad-spectrum initiator is one or a mixture of several broad-spectrum initiating substances, or a mixed system containing one or a mixture of several broad-spectrum initiating substances.

[0100] In some embodiments, the biomacromolecules include but are not limited to organic acids, polysaccharides and their derivatives, and the organic acids include but are not limited to lactic acid, tannic acid, citric acid, etc. In some embodiments, the polysaccharides and their derivatives include but are not limited to chitin, chitosan, etc.

[0101] In some embodiments, the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.

[0102] In some embodiments, the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, and the like.

[0103] In some embodiments, the antioxidants include but are not limited to vitamins such as niacinamide.

[0104] In some embodiments, the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.

[0105] In some embodiments, the metal ions include, but are not limited to, potassium, calcium, magnesium ions, and the like.

[0106] In some embodiments, the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.

[0107] In some embodiments, the positive charge source is urea or nicotinamide mononucleotide.

[0108] In some embodiments, the mixed system containing the positive charge source substance is serum, plasma, cell culture medium, animal and plant tissue fluid, etc., or a mixed solution containing the above positive charge source substances.

[0109] The cell culture medium includes but is not limited to: complete cell culture medium, animal-free cell culture medium, animal protein-free cell culture medium, and chemically defined cell culture medium.

[0110] In some embodiments, the drug is a substance having at least one of the following functions: hemostasis, anti-inflammation, antimicrobial, antifungal, antiviral, antimycoplasma, anticoagulant, analgesia, and promotion of cell, organ, and tissue growth and development.

[0111] As an initiator, the initiator includes but is not limited to gentamicin, kanamycin, etc.; the biochemical molecules of the positive electrochemical group are preferably selected from basic amino acids, nucleotides, nucleic acids, oligosaccharides, polysaccharides, vitamins, urea, peptides, peptoids, positively modified synthetic polymers, nanoparticles or microparticles, and cosmetics; the positively modified synthetic polymers include but are not limited to polylysine and polyarginine; the amino polysaccharides include but are not limited to chitosan; the basic amino acids include but are not limited to lysine and arginine; the functional molecules include but are not limited to spermine, spermidine, magnesium ions, and nicotinamide mononucleotide.

[0112] In some embodiments, the initiator is a mixture or mixed system comprising one or more of polylysine, polyarginine, spermine, spermidine, magnesium ion, gentamicin, kanamycin, arginine, lysine, chitosan, urea, and nicotinamide mononucleotide.

[0113] In some embodiments, the initiator is a mixed system of animal tissue fluid.

[0114] The three-dimensional mesh fiber structure formed by self-assembly can bind at least one of the following substances: biochemical molecules with positive electrochemical groups, drugs, functional molecules, animal tissue fluid, and cell culture storage or drug and functional molecule delivery systems containing the above components.

[0115] In some embodiments, the initiator can be dissolved in a solvent to obtain a solution, and the pH of the self-assembling peptide-initiator solution obtained by mixing the solution with the self-assembling peptide solution is 6.5 to 10, preferably 6.5-8.0, more preferably 6.5-7.5, and more preferably 7.0-7.5.

[0116] The principle of the self-assembling peptides of the present invention forming hydrogels in response to different triggering substances is consistent.

[0117] In a second aspect, the present invention provides a method for forming a scaffold material from the broad-spectrum responsive self-assembling peptide of the first aspect, the method comprising the step of initiating the self-assembling peptide to form a scaffold material with a broad-spectrum initiator.

[0118] In some embodiments, the broad-spectrum initiator is a positive charge source substance or a mixed system comprising a positive charge source substance, wherein the positive charge source substance includes a substance with a positively charged group or a positively charged ion.

[0119] In some embodiments, the positive charge source material is a biomacromolecule, drug, functional molecule, animal tissue fluid, or a cell culture storage or drug and functional molecule delivery system containing the above components, whose number of hydrogen bond acceptors is less than the number of hydrogen bond donors under neutral conditions.

[0120] In some embodiments, the broad-spectrum triggering substance is selected from physiological molecules, biochemical molecules, drugs, functional molecules, metal ions, microparticles or microspheres with positively charged surfaces containing positive electrochemical groups, or a mixed system of one or more thereof, or a mixed system of one or more of animal tissue fluid, complete cell culture medium, serum-free culture medium, cell culture storage fluid, or drug and functional molecule delivery systems containing the aforementioned substances.

[0121] In some embodiments, the broad-spectrum initiator is one or a mixture of several broad-spectrum initiating substances, or a mixed system containing one or a mixture of several broad-spectrum initiating substances.

[0122] In some embodiments, the biomacromolecules include but are not limited to organic acids, polysaccharides and their derivatives, and the organic acids include but are not limited to lactic acid, tannic acid, citric acid, etc. In some embodiments, the polysaccharides and their derivatives include but are not limited to chitin, chitosan, etc.

[0123] In some embodiments, the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.

[0124] In some embodiments, the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, and the like.

[0125] In some embodiments, the antioxidants include but are not limited to vitamins such as niacinamide.

[0126] In some embodiments, the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.

[0127] In some embodiments, the metal ions include, but are not limited to, potassium, calcium, magnesium ions, and the like.

[0128] In some embodiments, the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.

[0129] In some embodiments, the positive charge source is urea or nicotinamide mononucleotide.

[0130] In some embodiments, the mixed system containing the positive charge source substance is a cell culture medium, animal or plant tissue fluid, or a mixed solution containing the above-mentioned positive charge source substance.

[0131] The cell culture medium includes but is not limited to: complete cell culture medium, animal-free cell culture medium, animal protein-free cell culture medium, and chemically defined cell culture medium.

[0132] In some embodiments, the drug is a substance having at least one of the following functions: hemostasis, anti-inflammation, antimicrobial, antifungal, antiviral, antimycoplasma, anticoagulant, analgesia, and promotion of cell, organ, and tissue growth and development.

[0133] As an initiator, the initiator includes but is not limited to gentamicin, kanamycin, etc.; the biochemical molecules of the positive electrochemical group are preferably selected from basic amino acids, nucleotides, nucleic acids, oligosaccharides, polysaccharides, vitamins, urea, peptides, peptoids, positively modified synthetic polymers, nanoparticles or microparticles, poly (L-lactic acid) microspheres or polycaprolactone microspheres; the positively modified synthetic polymers include but are not limited to polylysine and polyarginine; the amino polysaccharides include but are not limited to chitosan; the basic amino acids include but are not limited to lysine and arginine; the functional molecules include but are not limited to spermine, spermidine, magnesium ions, and nicotinamide mononucleotide.

[0134] In some embodiments, the initiator is a mixture or mixed system comprising one or more of polylysine, polyarginine, spermine, spermidine, magnesium ion, gentamicin, kanamycin, arginine, lysine, chitosan, urea, and nicotinamide mononucleotide.

[0135] In some embodiments, the initiator is a mixed system of animal tissue fluid.

[0136] The three-dimensional mesh fiber structure formed by self-assembly can bind at least one of the following substances: biochemical molecules with positive electrochemical groups, drugs, functional molecules, animal tissue fluid, and cell culture storage or drug and functional molecule delivery systems containing the above components.

[0137] In some embodiments, the initiator can be dissolved in a solvent to obtain a solution, and the pH of the self-assembling peptide-initiator solution obtained by mixing the solution with the self-assembling peptide solution is 6.5 to 10, preferably 6.5-8.0, more preferably 6.5-7.5, and more preferably 7.0-7.5.

[0138] The principle of the self-assembling peptides of the present invention forming hydrogels in response to different triggering substances is consistent.

[0139] The solution self-assembling peptide can be dissolved in a neutral or alkaline solvent, and the solution can be adjusted after dissolution. The solvent includes one or more aqueous solutions of sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonia water, etc. that can provide an alkaline environment.

[0140] The solvent can be used to dissolve the self-assembling peptide of the present invention as long as it can provide a neutral or alkaline environment solution, preferably a physiologically acceptable solution.

[0141] In some embodiments, under the conditions of the mixed system of the broad-spectrum responsive self-assembling peptides, the polypeptides respond and self-assemble into a hydrogel within 120 minutes, preferably 60 minutes, and more preferably within 10 minutes to form the scaffold material.

[0142] In some embodiments, the method can also regulate the strength of the hydrogel material, the mechanical properties of the self-assembling peptide hydrogel, the storage modulus value of the hydrogel, and the formation time of the hydrogel by adjusting the type, composition, and concentration of the initiating substance.

[0143] In the mixed system of the acidic amino acids of the self-assembling peptide and the broad-spectrum initiator, the broad-spectrum initiating substance interacts with the self-assembling peptide to induce the peptide aqueous solution to self-assemble into a scaffold material in the form of a hydrogel.

[0144] In particular, under physiological conditions, such as neutral physiological conditions, a broad-spectrum initiating substance or a mixed system containing a broad-spectrum initiating substance is added to provide a positive charge, thereby neutralizing the negatively charged acid ions on the self-assembling peptide molecules, thereby reducing the repulsive force between the self-assembling peptide molecules. The self-assembling peptide molecules then achieve self-assembly through hydrophobic interactions and hydrogen bonds, ultimately forming a three-dimensional network nanostructure.

[0145] In some embodiments, the three-dimensional mesh scaffold material is in the form of a hydrogel or a dry form of a hydrogel, such as a freeze-dried powder of a hydrogel.

[0146] In the hydrogel, the concentration of the broad-spectrum responsive self-assembling peptide is above 0.1 wt.%, more preferably within the range of 0.15-5 wt.%, and even more preferably within the range of 0.2-1 wt.%.

[0147] The multi-responsive self-assembling peptides of the present invention respond rapidly to triggering substances. In the presence of triggering substances, the multi-responsive peptides can self-assemble into a three-dimensional network scaffold within a relatively short period of time, achieving gelation. The time it takes for the hydrogel to form is affected by the type, composition, and concentration of the triggering substance (relative concentration to the self-assembling peptide). The self-assembling peptides respond by self-assembling into a hydrogel within 120 minutes, preferably 60 minutes, and more preferably 10 minutes, of contact with the triggering substance.

[0148] In a third aspect, the present invention provides a method for regulating the mechanical properties of a self-assembling peptide hydrogel material, or regulating the formation time of a hydrogel, the method comprising the steps of adjusting the type, composition and / or concentration of a triggering substance.

[0149] In some embodiments, adjusting the concentration of the triggering substance is adjusting the relative concentration of the triggering substance and the self-assembling peptide.

[0150] In some embodiments, the method further comprises the step of adjusting the concentration of the self-assembling peptide.

[0151] The initiator's effect on the self-assembly of the self-assembling peptide is primarily influenced by electrostatic interactions. The charge concentration ratio of the initiator to the self-assembling peptide is 1:100-100:1. The dosage of the self-assembling peptide should be kept as low as possible to achieve the desired effect, thus saving material costs. Customization of the nanomaterial can be achieved by combining different initiators or varying the ratio of initiator to self-assembling peptide.

[0152] In the controllable multi-responsive self-assembling peptide hydrogel material of the present invention, the concentration of the self-assembling peptide is preferably above 0.1 wt.%, more preferably in the range of 0.15-5 wt.%, and even more preferably in the range of 0.2-1 wt.%.

[0153] In a fourth aspect, the present invention provides a three-dimensional network scaffold material in the form of a hydrogel, wherein the three-dimensional network scaffold material comprises the broad-spectrum responsive self-assembling peptide of the first aspect.

[0154] In some embodiments, the three-dimensional mesh scaffold material is obtained by the method of the second aspect or the third aspect.

[0155] In some embodiments, the three-dimensional mesh scaffold material is in the form of a hydrogel or a dry form of a hydrogel, such as a freeze-dried powder of a hydrogel.

[0156] In some embodiments, the three-dimensional mesh scaffold material is in the form of an injectable hydrogel.

[0157] In some embodiments, the three-dimensional mesh scaffold material is a nanostructure.

[0158] In the hydrogel, the concentration of the broad-spectrum responsive self-assembling peptide is above 0.1 wt.%, more preferably in the range of 0.15-5 wt.%, and even more preferably in the range of 0.2-1 wt.%.

[0159] The three-dimensional network scaffold material has more β-sheet structures than the self-assembling peptide.

[0160] In a fifth aspect, the present invention provides a composition comprising the broad-spectrum responsive self-assembling peptide of the first aspect and a broad-spectrum initiator.

[0161] The composition is in the form of being combined together, or in the form of being combined, wherein the latter is that the self-assembling peptide and the broad-spectrum initiator are placed in different containers respectively.

[0162] In some embodiments, the broad-spectrum initiator is a positive charge source substance or a mixed system comprising a positive charge source substance, wherein the positive charge source substance includes a substance with a positively charged group or a positively charged ion.

[0163] In some embodiments, the positive charge source material is a biomacromolecule, drug, functional molecule, animal tissue fluid, or a cell culture storage or drug and functional molecule delivery system containing the above components, whose number of hydrogen bond acceptors is less than the number of hydrogen bond donors under neutral conditions.

[0164] In some embodiments, the broad-spectrum triggering substance is selected from physiological molecules, biochemical molecules, drugs, functional molecules, metal ions, microparticles or microspheres with positively charged surfaces containing positive electrochemical groups, or a mixed system of one or more thereof, or a mixed system of one or more of animal tissue fluid, complete cell culture medium, serum-free culture medium, cell culture storage fluid, or drug and functional molecule delivery systems containing the aforementioned substances.

[0165] In some embodiments, the broad-spectrum initiator is one or a mixture of several broad-spectrum initiating substances, or a mixed system containing one or a mixture of several broad-spectrum initiating substances.

[0166] In some embodiments, the biomacromolecules include but are not limited to organic acids, polysaccharides and their derivatives, and the organic acids include but are not limited to lactic acid, tannic acid, citric acid, etc. In some embodiments, the polysaccharides and their derivatives include but are not limited to chitin, chitosan, etc.

[0167] In some embodiments, the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.

[0168] In some embodiments, the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, and the like.

[0169] In some embodiments, the antioxidants include but are not limited to vitamins such as niacinamide.

[0170] In some embodiments, the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.

[0171] In some embodiments, the metal ions include, but are not limited to, sodium, potassium, calcium, magnesium ions, and the like.

[0172] In some embodiments, the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.

[0173] In some embodiments, the positive charge source is urea or nicotinamide mononucleotide.

[0174] In some embodiments, the mixed system containing the positive charge source substance is serum, plasma, cell culture medium, animal and plant tissue fluid, etc., or a mixed solution containing the above positive charge source substances.

[0175] The cell culture medium includes but is not limited to: complete cell culture medium, animal-free cell culture medium, animal protein-free cell culture medium, and chemically defined cell culture medium.

[0176] In some embodiments, the drug is a substance having at least one of the following functions: hemostasis, anti-inflammation, antimicrobial, antifungal, antiviral, antimycoplasma, anticoagulant, analgesia, and promotion of cell, organ, and tissue growth and development.

[0177] As an initiator, the initiator includes but is not limited to gentamicin, kanamycin, etc.; the biochemical molecules of the positive electrochemical group are preferably selected from basic amino acids, nucleotides, nucleic acids, oligosaccharides, polysaccharides, vitamins, urea, peptides, peptoids, positively modified synthetic polymers, nanoparticles or microparticles, and cosmetics; the positively modified synthetic polymers include but are not limited to polylysine and polyarginine; the amino polysaccharides include but are not limited to chitosan; the basic amino acids include but are not limited to lysine and arginine; the functional molecules include but are not limited to spermine, spermidine, magnesium ions, and nicotinamide mononucleotide.

[0178] In some embodiments, the initiator is a mixture or mixed system comprising one or more of polylysine, polyarginine, spermine, spermidine, magnesium ion, gentamicin, kanamycin, arginine, lysine, chitosan, urea, and nicotinamide mononucleotide.

[0179] In some embodiments, the initiator is a mixed system of animal tissue fluid.

[0180] In the hydrogel, the concentration of the broad-spectrum responsive self-assembling peptide is above 0.1 wt.%, more preferably within the range of 0.15-5 wt.%, and even more preferably within the range of 0.2-1 wt.%.

[0181] The multi-responsive self-assembling peptides of the present invention respond rapidly to triggering substances. In the presence of triggering substances, the multi-responsive peptides can self-assemble into a three-dimensional network scaffold within a relatively short period of time, achieving gelation. The time it takes for the hydrogel to form is affected by the type, composition, and concentration of the triggering substance (relative concentration to the self-assembling peptide). The self-assembling peptides respond by self-assembling into a hydrogel within 120 minutes, preferably 60 minutes, and more preferably 10 minutes, of contact with the triggering substance.

[0182] The present invention unexpectedly discovered that under the conditions of multiple initiators, the three-dimensional network scaffold structure formed is more stable. Therefore, such self-assembling peptides are particularly suitable for in vivo applications and have unparalleled advantages over other self-assembling peptides in the prior art. The three-dimensional network scaffold material is a nanostructure.

[0183] The resulting hydrogel also exhibits self-repair capabilities. Mechanical forces can disrupt the hydrogel's gel state and restore it to its gel state after the force is removed. This recovery time is no more than 10 minutes, and the hydrogel's storage modulus after recovery is at least 70%, preferably at least 85%, and more preferably at least 95% of its pre-destruction value.

[0184] In a sixth aspect, the present invention provides the application of the broad-spectrum responsive self-assembling peptide of the first aspect, the method of the second aspect or the third aspect, the three-dimensional mesh scaffold material of the fourth aspect or the composition of the fifth aspect in one or more selected from the following: regenerative medicine and tissue regeneration; 2D and 3D cell culture and storage; dispersion and embedding filling of microspheres; dispersion and support of microcarriers in cell 3D culture systems; drug delivery; wound healing; implantable materials; gene therapy; stem cell therapy; and medical cosmetology.

[0185] The hydrogel of the present invention is safe and convenient to prepare, and does not require adjustment of the pH value, temperature, light, salt or ion components of the system. Common initiating substances in the biomedical field can trigger self-assembly to form a gel, thereby better ensuring the biocompatibility of the hydrogel of the present invention.

[0186] The broad-spectrum responsive self-assembling peptides provided by the present invention, as well as the hydrogels prepared therefrom, can be used for in vitro three-dimensional culture and storage, establishing cell models, loading cells, organs, or organoids, and injecting them into animals or humans for tissue repair. They can also be used as wound dressings, hemostatic materials, etc., or as carriers for sustained-release drugs or functional factors, or as cell preservation materials in biotherapy, tissue engineering, and regenerative medicine. In summary, the broad-spectrum responsive self-assembling peptides of the present invention, as well as the hydrogels prepared therefrom, have a wide range of applications and are safe and convenient.

[0187] The strength of the interaction between different triggering substances and the acidic amino acids of the self-assembling peptide is the basis for the controllable self-assembling peptide hydrogel of the present invention. Its mechanical properties and functionality can be adjusted to varying degrees by changing the concentration of the self-assembling peptide and the triggering substance, thereby achieving customization and / or programming of the nanomaterial, making the hydrogel of the present invention more widely applicable to various scenarios.

[0188] The present invention provides a convenient and safe method for preparing a controllable broad-spectrum responsive self-assembling peptide hydrogel under physiological conditions, which is suitable for scenes such as laboratories, hospitals, and even the field, and is safe and highly operable. The method comprises: preparing the broad-spectrum responsive self-assembling peptide solution, adding or injecting it into an environment containing at least one of the triggering substances, and forming the broad-spectrum responsive self-assembling peptide hydrogel under neutral or physiological conditions or in an animal body. The preparation method is simple and fast, and does not require adjusting the pH value, temperature, light, salt or ion components of the system. The broad-spectrum responsive self-assembling peptide hydrogel of the present invention can be formed in situ within half an hour, and the operability is strong. In particular, the broad-spectrum responsive self-assembling peptide of the present invention can be triggered to gel by endogenous substances, such as common substances in the biomedical field such as tissue fluid, complete cell culture medium, serum-free cell culture medium, and some artificially synthesized drugs, which reduces the requirements of the self-assembling peptide gelation process for application scenarios such as cell culture and storage, tissue filling and repair, and better ensures the biocompatibility of the broad-spectrum responsive self-assembling peptide hydrogel of the present invention.

[0189] The above-mentioned adjustable broad-spectrum responsive self-assembling peptide hydrogel material of the present invention can be applied to 3D cell culture and storage, tissue engineering, regenerative medicine, drug delivery and other aspects, and has a wide range of applications. For example, the adjustable broad-spectrum responsive self-assembling peptide hydrogel of the present invention can be used to culture different types of cells, realize in vitro three-dimensional culture, and establish a cell model; the adjustable broad-spectrum responsive self-assembling peptide hydrogel of the present invention loaded with cells / organoids / organs can be injected into animals for in vitro 3D culture and tissue repair and other research and applications; or the self-assembling peptide solution of the present invention is directly injected to form a self-assembling peptide hydrogel with the tissue fluid of the animal / human body, which is used as a wound dressing, hemostatic material, etc.; or after the drug / functional molecule is mixed with the self-assembling peptide solution of the present invention to form a self-assembling peptide hydrogel, it is injected or applied to a wound or lesion in vitro or in vivo as a carrier for sustained release of drugs or functional factors; or the adjustable broad-spectrum responsive self-assembling peptide hydrogel of the present invention is mixed with a cell suspension for cell preservation. The adjustable broad-spectrum responsive self-assembling peptide hydrogel of the present invention has a wide range of applications and is safe, and does not introduce risk substances into the application scenario. BRIEF DESCRIPTION OF THE DRAWINGS

[0190] Figures 1A-1B show macroscopic images of a self-assembling peptide solution and a mixture of a self-assembling peptide and an initiating substance according to the present invention. Figure A is a 0.5 wt.% self-assembling peptide solution; Figure B is a mixture of a self-assembling peptide and arginine at a 0.5 wt.% peptide concentration. Figure 1C demonstrates that the self-assembling peptide can form a hydrogel using tissue fluid as an initiator, which remains in a gel state after being extruded through a syringe.

[0191] Figures 2A-2J are FESEM images of the self-assembling peptides of the present invention and mixtures of the self-assembling peptides and initiating substances, with a scale of 500 nm. Figure 2A shows the self-assembling peptide alone, Figure 2B shows a mixture of the self-assembling peptide and polylysine, Figure 2C shows a mixture of the self-assembling peptide and spermine, Figure 2D shows a mixture of the self-assembling peptide and gentamicin, Figure 2E shows a mixture of the self-assembling peptide and lysine, Figure 2F shows a mixture of the self-assembling peptide and arginine, Figure 2G shows a mixture of the self-assembling peptide and spermidine, Figure 2H shows a mixture of the self-assembling peptide and kanamycin, Figure 2I shows a mixture of the self-assembling peptide and chitosan, and Figure 2J shows a mixture of the self-assembling peptide and magnesium ions.

[0192] Figures 3A-3J are TEM images of the self-assembling peptides and mixtures of the self-assembling peptides and initiating substances of the present invention. Figure 3A shows only the self-assembling peptide, Figure 3B shows a mixture of the self-assembling peptide and polylysine, Figure 3C shows a mixture of the self-assembling peptide and spermine, Figure 3D shows a mixture of the self-assembling peptide and gentamicin, Figure 3E shows a mixture of the self-assembling peptide and lysine, Figure 3F shows a mixture of the self-assembling peptide and arginine, Figure 3G shows a mixture of the self-assembling peptide and spermidine, Figure 3H shows a mixture of the self-assembling peptide and kanamycin, Figure 3I shows a mixture of the self-assembling peptide and chitosan, and Figure 3J shows a mixture of the self-assembling peptide and magnesium ions.

[0193] FIG4 is a circular dichroism spectrum of the self-assembling peptide of the present invention and a mixture of the self-assembling peptide and an initiating substance.

[0194] FIG5 is a comparison of the mechanical properties of the self-assembled peptide of the present invention and mixtures of the self-assembled peptide with different types of initiating substances.

[0195] FIG6 shows the oscillation time scan results of the self-assembled peptide of the present invention and a mixture of the self-assembled peptide and an initiating substance (polylysine, spermine, gentamicin, lysine, arginine, chitosan, polyarginine, magnesium ion, urea, and nicotinamide mononucleotide, respectively).

[0196] FIG7 shows the results of shear thinning and self-repair experiments of the self-loaded peptide hydrogel of the present invention.

[0197] FIG8 shows the storage modulus G' and loss modulus G" of the self-loaded peptide hydrogel of the present invention after dilution 10 times.

[0198] FIG9 shows the changes in rheological properties of self-assembling peptides with different sequence structures.

[0199] Figure 10 is a schematic diagram of the self-assembling peptides of the present invention forming a hydrogel in response to tissue fluid. Bottle (A) contains tissue fluid, bottle (B) contains a 0.3 wt.% self-assembling peptide solution, and bottle (C) contains a mixture of the self-assembling peptide and tissue fluid with a final peptide concentration of 0.3 wt.%.

[0200] Figures 11A-11D show the dynamic rheological experimental results of the self-installed peptide of the present invention in response to common liquid environments in the body to form hydrogels. Figure 11A shows the changes in the storage modulus G' and loss modulus G" over time during the process of forming hydrogels in response to serum-free culture medium with a final peptide concentration of 0.5 wt.% of the self-installed peptide of the present invention; Figure 11B shows the changes in the storage modulus G' and loss modulus G" over time during the process of forming hydrogels in response to tissue fluid with a final peptide concentration of 0.5 wt.% of the self-installed peptide of the present invention; Figure 11C shows the stability of the self-installed peptide hydrogel of the present invention when tissue fluid is used as the triggering substance; Figure 11D shows the comparison of the modulus of the self-installed peptide hydrogel formed by the self-installed peptide of the present invention and that formed by the self-installed peptide hydrogel in response to tissue fluid.

[0201] Figure 12 shows the effect of the self-loading peptide hydrogel of the present invention on supporting red blood cells, wherein the triggering substance in tube (A) is kanamycin, the triggering substance in tube (B) is spermine, and the triggering substance in tube (C) is arginine.

[0202] Figures 13A-13D are confocal laser scanning microscopy images of the supporting effect of the self-loaded peptide hydrogel of the present invention on liver cancer cells: A is the control group, without the addition of the peptide of the present invention or the triggering substance; B and D are the distribution of liver cancer cells in the self-loaded peptide hydrogel of the present invention: the triggering substance of B is arginine, the triggering substance of C is spermidine, and the triggering substance of D is magnesium ions.

[0203] FIG14 shows the results of cell activity analysis when the self-loading peptide hydrogel of the present invention is used for three-dimensional cell culture.

[0204] FIG. 15 shows the cell viability of muscle satellite cells after three-dimensional culture in the peptide hydrogel of the present invention for 3 days.

[0205] Figures 16A-16I show bright field microscopic images and statistical graphs of cell sphere diameters of muscle satellite cells cultured three-dimensionally in the self-assembled peptide hydrogels of the present invention for 7 days. A shows cells cultured for 3 days with 0.1 wt.% SEQ ID NO: 3; B shows cells cultured for 7 days with 0.1 wt.% SEQ ID NO: 3; C shows the distribution of cell sphere diameters over 7 days with 0.1 wt.% SEQ ID NO: 3; D shows cells cultured for 3 days with 0.3 wt.% SEQ ID NO: 3; E shows cells cultured for 7 days with 0.3 wt.% SEQ ID NO: 3; F shows the distribution of cell sphere diameters over 7 days with 0.3 wt.% SEQ ID NO: 3; G shows cells cultured for 3 days with 0.5 wt.% SEQ ID NO: 3; H shows cells cultured for 7 days with 0.5 wt.% SEQ ID NO: 3; and I shows the distribution of cell sphere diameters over 7 days with 0.5 wt.% SEQ ID NO: 5.

[0206] 17A-17D are confocal laser scanning microscopy images of human umbilical cord-derived mesenchymal stem cells cultured three-dimensionally in the self-assembled peptide hydrogel of the present invention for 4 days (A, C) and 7 days (B, D).

[0207] FIG18 shows the effects of not (2D) and using (3D) the self-assembling peptide scaffold solution on the cell survival of mouse mesenchymal stem cells during refrigerated storage.

[0208] Figures 19A-19E show the suspension support effect of self-assembling peptides on microcarriers and the proliferation culture of mouse mesenchymal stem cells using this system, wherein A is a polystyrene microcarrier supported by a self-assembling peptide of SEQ ID NO: 25, B is a macroporous gelatin microcarrier supported by a self-assembling peptide of SEQ ID NO: 19, C is a polylactic acid microsphere supported by a self-assembling peptide of SEQ ID NO: 26, D is a fluorescence microscopy image under static culture conditions, and E is a cell counting result.

[0209] Figures 20A-20C demonstrate that a self-assembling peptide solution can stably disperse poly(L-lactic acid) microspheres. Figure A shows that the microspheres precipitate in an aqueous solution but are uniformly suspended and dispersed after mixing with the self-assembling peptide solution. Figure B shows that the self-assembling peptide mixture containing microspheres is liquid. Figure C shows that after mixing the self-assembling peptide mixture containing microspheres obtained in Figure B with tissue fluid, the self-assembling peptide / poly(L-lactic acid) microsphere solution rapidly forms a hydrogel.

[0210] Figures 21A-21C demonstrate the stable dispersion of polycaprolactone using a self-assembling peptide solution. Figure A shows that the microspheres precipitate in an aqueous solution but are uniformly suspended and dispersed after mixing with the self-assembling peptide solution. Figure B shows that the self-assembling peptide mixture containing microspheres is liquid. Figure C shows that after mixing the self-assembling peptide mixture containing microspheres obtained in Figure B with tissue fluid, the self-assembling peptide / L-lactic acid microsphere solution rapidly forms a hydrogel. DETAILED DESCRIPTION

[0211] The present invention obtains a self-assembling peptide by cleverly controlling the secondary structure of the polypeptide, uniquely selecting hydrophobic amino acids in the hydrophobic domain, uniquely selecting hydrophilic amino acids in the hydrophilic domain, and balancing and precisely coordinating the hydrophobic and hydrophilic amino acids.

[0212] The present invention provides a broad-spectrum responsive self-assembling peptide and also provides a hydrogel prepared from the broad-spectrum responsive self-assembling peptide in the presence of a positive charge source substance or a mixed system containing the positive charge source substance. The scaffold material in the form of the hydrogel is a hydrogel material having a three-dimensional network scaffold structure.

[0213] The broad-spectrum self-assembling peptides of the present invention are in a neutral liquid state during use under human physiological conditions, thus avoiding the risks caused by adjusting pH or introducing other exogenous substances, such as certain metal salt ions, specific proteins, etc.

[0214] The broad-spectrum self-assembling peptides of the present invention have a stronger support effect due to the presence of hydroxyproline (O) in the β-turn motif of the hydrophilic domain. In addition, the present invention has found that the presence of hydroxyproline (O) compared to proline (P) in the β-turn motif of the hydrophilic domain, the support effect of the self-assembling peptide is stronger.

[0215] The interaction between the acidic amino acids of the self-assembling peptides of the present invention and the initiator triggers the self-assembly of the peptide aqueous solution into a peptide hydrogel. Under neutral conditions, the addition of the initiator produces a positively charged substance, which neutralizes the negatively charged acid ions on the polypeptide molecules, thereby reducing the repulsive forces between the polypeptide molecules. The polypeptide molecules then self-assemble through hydrophobic interactions and hydrogen bonds, ultimately forming a nanoscale three-dimensional network structure.

[0216] The present invention provides a controllable broad-spectrum responsive self-assembling peptide hydrogel and a preparation and application method thereof. The controllable broad-spectrum responsive self-assembling peptide hydrogel contains an initiating substance and a self-assembling peptide that self-assembles into a three-dimensional network scaffold. The broad-spectrum responsive self-assembling peptide hydrogel material has shear-thinning properties and self-repairing properties, that is, the broad-spectrum responsive self-assembling peptide of the present invention and the broad-spectrum responsive self-assembling peptide hydrogel of the present invention are injectable. Moreover, because the tissue fluid itself can be used as an initiating substance, in situ gelation can be achieved by injecting the broad-spectrum responsive self-assembling peptide of the present invention. Therefore, the broad-spectrum responsive self-assembling peptide hydrogel of the present invention can be widely used in the biomedical field, including scaffold materials for tissue engineering, three-dimensional cell culture materials, drug delivery carriers, wound dressings, etc.

[0217] The broad-spectrum responsive self-assembling peptide hydrogel of the present invention has a broad-spectrum initiation, which is reflected in the ability to be triggered by a variety of different substances. Based on this, the self-assembling peptide of the present invention can respond to endogenous substances to form hydrogels, thereby avoiding changes in the physical and chemical components in the application environment. For some experiments with strict conditions, such as immune response experiments, suitable triggering substances can also be selected as needed to avoid creating false experimental results. Certain cell culture experiments require the use of serum-free culture medium. The self-assembling peptide of the present invention can respond to a variety of substances and can self-assemble into hydrogels in these systems to achieve three-dimensional cell culture. The controllable broad-spectrum responsive self-assembling peptide hydrogel of the present invention can be used in the fields of cell culture, tissue repair, drug delivery, etc. The gelation process does not require changes in ambient temperature and pH, does not rely on expensive precision instruments, does not require technical personnel to learn complicated operating procedures, and does not introduce any exogenous substances to bring unnecessary risks.

[0218] The presence of the triggering substance under physiological conditions (pH greater than 6.5, temperature 37°C) promotes the self-assembly of the broad-spectrum responsive self-assembling peptide of the present invention into a three-dimensional network scaffold system to form a broad-spectrum responsive self-assembling peptide hydrogel. The storage modulus of the hydrogel is not less than 20Pa. By changing the type, composition, and concentration of the triggering substance (relative concentration to the broad-spectrum responsive self-assembling peptide), the strength of the hydrogel material can be adjusted to a specific value to meet the preset requirements. In multiple embodiments of the present invention, different types of triggering substances are used, and the response degree of the broad-spectrum responsive self-assembling peptide is different, forming a differentiated nanostructure, and obtaining a controllable broad-spectrum responsive self-assembling peptide hydrogel. For the same triggering substance, changing its concentration can affect the ratio of the triggering substance to the self-assembling peptide, resulting in different mechanical properties of the obtained polypeptide hydrogel. Therefore, by adjusting the type and concentration of the triggering substance, the desired self-assembling peptide hydrogel material can be obtained in accordance with different application scenarios, realizing the personalized design of nanomaterials. For example, self-assembling peptide hydrogels with different supporting capabilities are obtained according to the optimal external growth environment of different cell lines.

[0219] The self-assembling peptide hydrogel has the properties of shear thinning and self-repair. The hydrogel structure will be temporarily destroyed by strong mechanical force. When the mechanical force disappears, the three-dimensional network scaffold structure inside the hydrogel will be rapidly reorganized to reform the hydrogel. The storage modulus will be restored to at least 60% before the gel is destroyed, and the self-repair time of the hydrogel is less than 10 minutes. Shear thinning can be carried out using a variety of mechanical forces that can apply shear or shear stress to the hydrogel, such as blowing, centrifugation, shaking, injection, spraying, filtration, etc. The polypeptide hydrogel still has the ability to self-repair after suffering multiple damages. Therefore, the self-assembling peptide hydrogel of the present invention is provided in an injectable form and can be gelled in situ after injection. In addition, the self-assembling peptide hydrogel of the present invention can be diluted to a liquid state, so that it cannot be reassembled into a hydrogel. When used as a cell culture material, it is beneficial to separate cells from the polypeptide hydrogel.

[0220] The self-assembling peptides disclosed herein may be a mixture of one or more self-assembling peptides. The self-assembling peptides comprise a relatively hydrophobic domain and a relatively hydrophilic hydrophilic domain. The relatively hydrophilic hydrophilic domain contains at least two consecutive β-turn motifs forming a β-turn structure, wherein at least one β-turn motif terminates with an acidic amino acid. The self-assembling peptides may be a mixture of one or more self-assembling peptides. The hydrophobic domain may also contain hydrophilic amino acids, and the hydrophilic domain may also contain hydrophobic amino acids.

[0221] The self-assembling peptide solution comprises at least one self-assembling peptide dissolved in a solvent system at a concentration higher than 0.1 wt.%. The self-assembling peptide can use a neutral or alkaline solution as a solubilizing agent, and after dissolution, the pH of the polypeptide solution is adjusted to 6.5 to 10.0, preferably 7.0-7.5. The solubilizing agent includes sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonia water, and the like, and mixtures thereof, which are dissolved in water and can provide an alkaline solution environment, or a neutral solvent such as water. It should be understood that only common solutions are listed here, but as long as a neutral or alkaline environment can be provided, the polypeptide can be dissolved, preferably a physiologically acceptable solution.

[0222] Because the self-assembling peptides described herein form hydrogels in response to different triggering substances using the same principle, various examples provide validation data for the properties and functions of the self-assembling peptides described herein, using different self-assembling peptide sequences as examples. It is understood that other polypeptides conforming to the polypeptide structures of the present invention, for which detailed data are not provided herein, may also have equivalent or similar effects.

[0223] The controllable multi-responsive self-assembling peptide hydrogel of the present invention is formed by the self-assembling peptide in response to a variety of triggering substances. The broad-spectrum responsiveness is reflected in the presence of a variety of triggering substances, including biochemical molecules containing positive electrochemical groups, drugs, functional molecules, animal tissue fluid (tissue fluid is different from blood, there is a barrier between the two, and the tissue fluid contains only a small amount of large protein molecules), and cell culture storage or drug and functional molecule delivery systems containing the above components; the triggering substance is one or more of the above substances and a mixed system containing the above substances.

[0224] The drug possesses at least one of the following functions: hemostasis, anti-inflammatory, antimicrobial, antifungal, antiviral, anti-mycoplasma, anticoagulant, analgesic, and promotion of cell, organ, or tissue growth and development. The triggering substance includes, but is not limited to, gentamicin and kanamycin. The biochemical molecule with the positively charged chemical group is preferably selected from basic amino acids, nucleotides, nucleic acids, oligosaccharides, polysaccharides, vitamins, urea, peptides, peptoids, positively charged synthetic polymers, nanoparticles or microparticles, and cosmetics. The positively charged synthetic polymer includes, but is not limited to, polylysine and polyarginine. The aminopolysaccharide includes, but is not limited to, chitosan. The basic amino acids include, but are not limited to, lysine and arginine. The functional molecule includes, but is not limited to, spermine, spermidine, magnesium ions, and nicotinamide mononucleotide.

[0225] The initiating substance comprises a mixture of a plurality of the above-mentioned substances. In some embodiments, the initiating substance is a mixture of the above-mentioned initiating substances, such as cell culture medium and animal tissue fluid.

[0226] The three-dimensional mesh fiber structure formed by self-assembly can bind at least one of the following substances: biochemical molecules with positive electrochemical groups, drugs, functional molecules, animal tissue fluid, and cell culture storage or drug and functional molecule delivery systems containing the above components.

[0227] In one or more embodiments, the initiating substance can be dissolved to obtain a solution, and the pH of the self-assembling peptide-initiating substance solution obtained by mixing the solution with the self-assembling peptide solution is 6.5 to 10, preferably 6.5-8.0, more preferably 6.5-7.5, and more preferably 7.0-7.5.

[0228] Because the principles of the self-assembling peptides described herein in response to different triggering substances to form hydrogels are consistent, several of these triggering substances are selected to illustrate the technical effects of the self-assembling peptide hydrogels of the present invention. In some embodiments, polylysine, polyarginine, spermine, spermidine, magnesium ions, gentamicin, kanamycin, arginine, lysine, chitosan, urea, nicotinamide mononucleotide, tissue fluid, complete cell culture medium, and serum-free culture medium are used as examples to provide validation data for the multi-responsive properties and functions of the self-assembling peptides of the present invention. It is understood that other triggering substances for which detailed data are not provided in this invention may also have equivalent or similar effects.

[0229] The self-assembling peptide hydrogel of the present invention is formed by self-assembling peptides in response to a triggering substance under neutral conditions. The principle is briefly described below:

[0230] The side chains of the self-assembling peptides of the present invention carry a negative charge. The electrostatic interaction between the initiating substance and the self-assembling peptide can neutralize some or all of the negative charges in the self-assembling peptide solution system, reduce intermolecular electrostatic repulsion, and promote self-assembly. The self-assembling peptides of the present invention cannot independently assemble into a hydrogel in the absence of an initiating substance under neutral conditions. The electrostatic repulsion between polypeptide molecules hinders the hydrophobic interaction between the hydrophobic ends, making it difficult to form a regular and compact molecular arrangement. The sequence segments of the β-turn structure make the acidic amino groups more active, further exerting their effect. The peptide molecules tend to form hydrogen bonds with water molecules, increasing the difficulty of the self-assembling peptides to self-assemble into a three-dimensional mesh scaffold structure. When the initiating substance is present, the carbonyl groups on the self-assembling peptides will generate electrostatic attraction with the initiating substance. The negative charges on the self-assembling peptide molecules are partially or completely "shielded," resulting in electrical neutrality. The intermolecular electrostatic repulsion is reduced, and the hydrophobic interaction and hydrogen bonding cause the molecules to tend to aggregate in an orderly manner and be distributed regularly. Furthermore, the interaction between the polar amino acids on the self-assembling peptides of the present invention and the initiating substance reduces the polarity of the self-assembling peptide molecules, affecting their hydrophilicity and thus decreasing their solubility, leading to the formation of a hydrogel structure. From a molecular conformational perspective, the initiating substance influences the self-assembly process of the self-assembling peptides to form highly ordered aggregates dominated by β-pleated structures, resulting in a stable, highly oriented fiber structure.

[0231] Since the initiating substance and the self-assembling peptide are connected by a non-covalent bond, the physicochemical properties of the initiating substance will not be affected by gelation. Changes in the type, composition, and concentration (relative concentration to the self-assembling peptide) of the initiating substance affect the molecular interaction between it and the self-assembling peptide, causing changes in the mechanical properties of the self-assembling peptide hydrogel on a macroscopic scale, which is intuitively manifested in the increase or decrease in the storage modulus value of the hydrogel. The self-assembly stimulation of the self-assembling peptide by the initiating substance is mainly affected by electrostatic interactions, and the charge concentration ratio of the initiating substance to the self-assembling peptide is 1:100-100:1. The amount of self-assembling peptide used should be as low as possible to achieve the desired effect in order to save material costs. Customization of nanomaterials can be achieved by combining different initiating substances or changing the ratio of initiating substances to self-assembling peptides.

[0232] In the controllable multi-responsive self-assembling peptide hydrogel material of the present invention, the concentration of the self-assembling peptide is preferably above 0.1 wt.%, more preferably in the range of 0.15-5 wt.%, and even more preferably in the range of 0.2-1 wt.%.

[0233] The multi-responsive self-assembling peptides of the present invention respond rapidly to triggering substances. In the presence of triggering substances, the multi-responsive peptides can self-assemble into a three-dimensional network scaffold within a relatively short period of time, achieving gelation. The time it takes for the hydrogel to form is affected by the type, composition, and concentration of the triggering substance (relative concentration to the self-assembling peptide). The self-assembling peptides respond by self-assembling into a hydrogel within 120 minutes, preferably 60 minutes, and more preferably 10 minutes, of contact with the triggering substance.

[0234] The preparation method of the controllable multi-responsive self-assembling peptide hydrogel of the present invention is simple and easy to operate. By mixing the self-assembling peptide solution with an initiating substance solution or adding the self-assembling peptide solution to an environment containing an initiating substance, a self-assembling peptide hydrogel material can be obtained. The preparation process does not require any other changes to the system, and there is no need to change or adjust the temperature or pH of the system, nor to introduce other specific chemical components or add certain substances (salt ions, specific proteins, etc.). The self-assembling peptide of the present invention forms a three-dimensional mesh scaffold material in the presence of a positive charge source material, has a broad spectrum, and does not need to or preferably does not change the chemical composition and environmental conditions of the system. The self-assembling peptide hydrogel of the present invention can achieve three-dimensional cell culture. The self-assembling peptide of the present invention is directly mixed with a cell culture fluid containing cells and used, and is transferred to a culture dish, a culture flask, a cell culture well plate or other culture equipment to achieve three-dimensional cell culture. The culture conditions are consistent with traditional two-dimensional cell culture. Because the cell culture medium contains initiating substances such as spermine, lysine, arginine, spermidine, magnesium ions, the self-assembling peptide can be stimulated to form a three-dimensional mesh scaffold material that is conducive to cell adhesion and proliferation. The present invention is applicable to various types of cells, including cells such as stem cells that have strict growth condition requirements.

[0235] The self-assembling peptide hydrogel material of the present invention is also suitable as a hemostatic composition or for promoting wound healing. It can be injected or applied to the wound site. The tissue fluid in the patient's body acts as a triggering substance, which can cause the self-assembling peptides to respond and form a peptide hydrogel material at the injection site or wound site, thereby playing a supporting role or promoting healing.

[0236] Currently used and widely studied peptide or protein hydrogels for cell culture require consideration of the hydrogel's initiation mechanism when applying materials. The self-assembling peptide hydrogel of the present invention can be formed by triggering substances to stimulate polypeptide self-assembly under physiological conditions (pH = 7.2-7.4, around 37°C). The triggering substances can be adjusted according to the usage scenario to minimize the impact of the initiation of self-assembly on the original system. It is responsive to a variety of common substances in the body and has a broad spectrum. In addition, the mechanical properties of the self-assembling peptide hydrogel of the present invention can be adjusted by simply changing the type, composition, and concentration of the triggering substance (relative concentration to the self-assembling peptide).

[0237] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0238] Glossary:

[0239] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0240] Hydrogel is a hydrophilic polymer material that can form a three-dimensional network structure through chemical or physical crosslinking. Hydrogel materials can come from natural and synthetic sources. Natural polymers such as chitosan, alginate, hyaluronic acid (HA), collagen, gelatin, etc. have the advantages of being biodegradable and carrying integrin binding sites, but they are immunogenic. Synthetic polymers such as polyethylene glycol (PEG), polyacrylamide (PAM), polyvinyl alcohol (PVA) and polymethyl methacrylate (PMMA) have the advantages of strong mechanical properties, customizability, and low immunogenicity, but lack inherent biological functions and must undergo important post-processing to induce the desired response in vivo.

[0241] Hydrogels have the following properties:

[0242] 1. Good biological relevance: The polymer contains a large number of hydrophilic groups, which can absorb water dozens of times more than its own amount, and has the characteristics of swelling but not dissolving in water, and has good water retention capacity;

[0243] 2. Similar to the extracellular matrix: Through structural design, the physical, chemical and mechanical properties of the hydrogel can be made similar to those of the extracellular matrix, which is conducive to cell growth and reproduction;

[0244] 3. Biodegradability: Some natural polymer materials are biodegradable, preventing secondary damage caused by implant removal. It is these unique advantages that make hydrogels shine in biomedical materials.

[0245] In the present invention, when a peptide having a structure having both a hydrophilic surface and a hydrophobic surface self-assembles under the conditions described in the present invention, especially physiological conditions, a hydrogel is obtained by encapsulating water.

[0246] Peptide Self Assembly is a short chain of amino acids with a polar domain. When dissolved in a neutral solvent and physiological salt concentration, these self-assembling peptides spontaneously assemble into hierarchical nanostructures through hydrogen bonds, ionic bonds, hydrophobic interactions or van der Waals forces. Materials derived from these assemblies have the advantages of being non-toxic, non-immunogenic, non-thrombogenic, degradable and easily metabolized. At the same time, nanofibers have the same size scale as natural ECM fibers and can be easily designed to mimic the stiffness of various soft tissues. They can also be further functionalized by the attachment of cell-interacting peptide domains or cytokines and growth factors. Therefore, they can be used to design biologically relevant culture environments and improve the control of proliferating cell populations.

[0247] Self-assembling peptides can be used as drug carriers in hydrogels for wound treatment and in self-assembling peptide nanofibers for cancer treatment, enabling the sustained release of small molecules, growth factors, and monoclonal antibodies. For example, self-assembling peptides can be used to promote angiogenesis in regenerating tissues and to repair skin wounds using self-assembling peptide-based scaffolds. However, the application of self-assembling peptides in injectable therapeutic hydrogels is still in its infancy.

[0248] The alternating hydrophilic and hydrophobic amino acid residues in the self-assembling peptides allow them to retain large amounts of water and thus form hydrogels. The side chains of the hydrophilic residues can directly interact with water, with water molecules forming inclusion complexes to surround the side chains of the hydrophobic residues. The number of hydrophobic and hydrophilic residues in the self-assembling peptides requires clever design and proportioning. If there are too many hydrophobic residues, the self-assembling peptide will be insoluble in water and precipitate out of the water; on the other hand, if there are too many hydrophilic residues, the self-assembling peptide will be highly water-soluble and therefore unable to form a hydrogel. In addition, it is also necessary to precisely and cleverly induce the peptide molecules to self-assemble into ordered nanostructured materials, such as nanofibers, nanotubes, and nanovesicles.

[0249] "Amino acid" includes those naturally occurring as well as non-naturally occurring amino acids, such as D-natural amino acids, beta and gamma derivatives. According to standard terminology, amino acid residue sequences can be named using three-letter or one-letter codes, for example: alanine (Ala, A); arginine (Arg, R); asparagine (Asp, N); aspartic acid (Aspartic acid, D), cysteine ​​(Cysteine, C); glutamine (Glutamine, Q); glutamic acid (Glutamic acid, E); glycine (Gly, G); histidine (His, H), isoleucine (Ile, I); leucine (Leu, L), lysine (Lysine, L), methionine (Met, M); phenylalanine (Ala, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W), tyrosine (Tyr, Y); valine (Val, V); selenocysteine ​​(Sec, U); hydroxyproline (Hyp, O).

[0250] In the present invention, a "peptide" is an amino acid chain. In particular, a peptide is 2 to 40 amino acids in length.

[0251] In the present invention, "self-assembly" refers to the aggregation of self-assembling peptides into an ordered structure under normal environmental conditions.

[0252] In the present invention, "β-fold" refers to a relatively extended periodic folded zigzag main chain conformation in a polypeptide chain, which is arranged in parallel or antiparallel, thereby forming a β-fold (sheet). The parallel or antiparallel conformation is determined based on the direction of the peptide arrangement from N to C terminus. Parallel arrangement means that the peptide chains are arranged from N to C terminus. Antiparallel arrangement means that the peptide chains are arranged in opposite directions (i.e., the first peptide chain is arranged from N to C terminus, and the opposite second peptide chain is arranged from C to N terminus). The parallel arrangement may include the two ends of the peptide being staggered with each other due to peptide translation. At least half of the length of the peptide is involved in the interaction force between peptides. In the antiparallel arrangement, the polypeptides are usually arranged in a line to provide two flush endpoints. This is a typical end-to-end complementary peptide.

[0253] A β-turn is an irregular secondary structure in proteins that causes a change in the direction of the polypeptide chain. β-turns often occur at the corner where the peptide chain makes a 180° turn. A β-turn consists of 3-5 amino acid residues, with the second residue being either proline (P) or hydroxyproline (O). A β-turn motif generally refers to a turn structure stabilized by hydrogen bonds between the carbonyl oxygen atom of the nth amino acid residue and the amide proton of the n+3th amino acid residue. β-turns, also known as β-elbows, reverse elbows, or β-loops, serve to connect β-strands.

[0254] The term "hydrophobicity" used in the present invention refers to a property of being inclined to repel water or being completely insoluble in water.

[0255] The term "hydrophilicity" in the present invention refers to the property of easily absorbing water and having a strong polar group that easily interacts with water.

[0256] Hydrophilic amino acids, also known as polar amino acids, have polar R groups that can generally form hydrogen bonds with water molecules, thus having a certain affinity for water molecules. Hydrophilic amino acids include: S, T, Y, C, U, N, Q, D, E, O, R, K, and H.

[0257] Hydrophobic amino acids, also known as non-polar amino acids, have non-polar R groups and have low or no affinity for water molecules, but have a high affinity for fat-soluble substances. They include: G, A, V, L, I, P, M, F, and W.

[0258] "Nanostructure" refers to structures with nanometer dimensions. Nanostructures can be any one-dimensional, two-dimensional, or three-dimensional shape, including nanofilms, nanofibers, nanorods, nanowires, nanofiber networks, nanospheres, nanohelices, and mixtures thereof. A nanostructure's surface has a one-dimensional structure at the nanoscale, meaning the surface thickness of the object is between 0.1 nm and 100 nm. Nanotubes have two nanometer dimensions, with diameters ranging from 0.1 to 100 nm and lengths potentially exceeding. Spherical nanoparticles have three nanometer dimensions, meaning the particle's size in each spatial dimension is between 0.1 and 100 nm.

[0259] Circular dichroism spectroscopy is the most widely used method for determining protein secondary structure and monitoring conformational changes of protein molecules induced by external conditions. Because the test is performed on liquids, the results obtained are closer to the secondary structure of proteins in real physiological environments. It is a fast, simple and relatively accurate method for studying protein conformation.

[0260] In the present invention, "response" and "initiation" are used to indicate that the self-assembling peptide forms a hydrogel in response to different initiators, or that different initiators are used to trigger the self-assembling peptide to form a hydrogel, and the two have the same meaning. In the present invention, initiator and initiating substance are used interchangeably and have the same meaning.

[0261] The following examples and accompanying drawings are provided to facilitate understanding of the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention.

[0262] Example 1: Synthesis of self-assembling peptides

[0263] The self-assembling peptide compound of the present invention is synthesized by a standard solid phase self-assembling peptide synthesis method. The amino acid sequence of the self-assembling peptide is shown in the sequence listing SEQ ID NO. 1-32.

[0264] Example 2: Preparation of self-assembling peptides

[0265] The self-assembling peptides of SEQ ID NOs. 1-32 of Example 1 were added to water or phosphate-buffered saline (PBS) buffer (pH = 7.2-7.4, unless otherwise specified, the PBS buffer used in the present invention is of this pH value). Alkali solution was added dropwise until the self-assembling peptides were completely dissolved. The solution was adjusted to a neutral pH of 7.2 to obtain a 5 wt.% self-assembling peptide mother solution. After autoclaving, the solution was stored at 4°C for future use. The mother solution was used to adjust the concentration of the self-assembling peptides to obtain a self-assembling peptide material of a predetermined concentration. For example, a certain amount of the self-assembling peptide mother solution was diluted with PBS buffer to obtain a 0.5 wt.% self-assembling peptide solution.

[0266] Example 3. Preparation of self-assembling peptide hydrogel

[0267] The self-assembling peptide solution obtained in Example 2 is evenly mixed with the initiating substance solution, and the charge concentration ratio of the self-assembling peptide to the initiating substance in the mixed solution is ensured to be (1-100): (1-100), thereby obtaining a self-assembling, three-dimensional network scaffold with multiple initiating substances. The pH value of the obtained mixed solution is neutral (about 6 to about 8, preferably about 6.5-7.5, preferably about 7-7.5).

[0268] Preparation of a mixed solution of self-assembling peptides and small molecule initiating substances: Taking lysine, arginine, gentamicin, kanamycin, spermine, spermidine, magnesium ions, and urea as examples, 0.08 wt.% lysine solution, arginine solution, gentamicin solution (calculated based on its 4 amino groups), kanamycin solution, urea solution, nicotinamide mononucleotide solution, 0.06 wt.% spermine solution, spermidine solution, and magnesium ion solution (the solvent in the above solutions is phosphate buffer with a neutral pH value) are taken and mixed evenly with the 1 wt.% self-assembling peptide solution obtained in Example 2 at a volume ratio of 1:1 to obtain a self-assembling peptide hydrogel material formed by self-assembly of the self-assembling peptide in response to the initiating substance. The concentration of the self-assembling peptide in the final solution is 0.5 wt.%, and the charge concentration ratio of the self-assembling peptide to the initiating substance is approximately 1:1.

[0269] For macromolecular initiating substances, polylysine, chitosan, and polyarginine are used as examples. For ease of calculation, a mixed solution is prepared in this embodiment at a mass fraction ratio of 1:1 between the macromolecule and the self-assembling peptide (ensuring a charge concentration of (1-100):(1-100)). Polylysine, chitosan, and polyarginine at a concentration of 1 wt.% (the solvent is phosphate buffer with a neutral pH value) are mixed with the 1 wt.% self-assembling peptide solution obtained in Example 2 at a volume ratio of 1:1 to obtain a self-assembling peptide hydrogel material formed by the self-assembling peptide in response to the initiating substance. The final concentration of the self-assembling peptide in the solution is 0.5 wt.%.

[0270] For initiators that are mixtures, a mixture of initiator and self-assembling peptide solution is generally prepared according to the desired final self-assembling peptide concentration. Mixing a 1 wt.% self-assembling peptide solution with an equal volume of initiator can yield the peptide hydrogel of the present invention with a self-assembling peptide concentration of 0.5 wt.%. The initiator can be surface-positively charged porous gelatin microspheres, tissue fluid, complete cell culture medium, or serum-free culture medium (animal-derived culture medium, animal protein-free culture medium, or chemically defined culture medium).

[0271] Taking the self-assembling peptide represented by the amino acid sequence IIIIGTVIGPGGEGOGGE (SEQ ID NO: 14) as an example, the initiating substance is arginine. A 0.5 wt.% peptide solution (see Figure 1A) is in liquid form. After the sample bottle is inverted, the solution flows back and collects at the bottle mouth. In the presence of arginine, a peptide hydrogel with a peptide concentration of 0.5 wt.% self-assembles in response to the initiating substance arginine (see Figure 1B). When the sample bottle is inverted, the material does not fall due to the hydrogel formation. Using tissue fluid as the initiator, the self-assembling peptide can form a hydrogel that remains in a gel state after being extruded through a syringe (see Figure 1C). This method also has the same or similar effects on peptide hydrogel materials formed in response to other types of initiating substances.

[0272] Taking the self-assembling peptide represented by the amino acid sequence LLLLLGSVLGPAGEGPAGE (SEQ ID NO: 29) as an example, a 2% concentration of the self-assembling peptide quickly formed a hydrogel after mixing with an equal volume of human tissue fluid. The hydrogel can be drawn up with a syringe and remains in a gel state after injection and extrusion. The peptide hydrogel materials formed in response to other types of triggering substances also have the same or similar effects.

[0273] According to this method, the self-assembling peptide hydrogel material with any predetermined peptide concentration and initiating substance concentration ratio described in the present invention can be prepared. The results are shown in Table 1.

[0274] Example 4. Characterization of peptide hydrogel materials

[0275] Experiment 1: Field Emission Scanning Electron Microscopy (FESEM)

[0276] The self-assembling peptide is exemplified by the amino acid sequence SEQ ID NO: 14 (IIIIIGTVIGPGGEGOGGE); the initiating substances are exemplified by polylysine, spermine, gentamicin, lysine, arginine, spermidine, kanamycin, chitosan, and magnesium ions.

[0277] Experimental method: The self-assembling peptide mother solution obtained in Example 2 (the self-assembling peptide shown in SEQ ID NO: 14) and the peptide hydrogel material formed by self-assembly of the self-assembling peptide in Example 3 in response to the triggering substance were diluted with ultrapure water. In order to obtain the nanoscale morphology of the hydrogel under physiological conditions that are more in line with the application scenario, they were incubated at 37°C for 1 hour to simulate the physiological environment of the cell. 10 μL was placed on a silicon wafer and allowed to stand and dry at room temperature. The surface morphology of the self-assembling peptide material shown in SEQ ID NO: 14 and the peptide hydrogel material formed by the self-assembling peptide in response to the triggering substance were observed using a field emission scanning electron microscope (FEI-Apreo) at 1 kilovolt. Before observation, gold was sprayed on the sample surface to increase the conductivity of the sample. The observation results are shown in Figures 2A-2J.

[0278] Experimental results: The self-assembled peptides of the present invention can assemble into amorphous fibrous nanostructures (Figure 2A) under neutral conditions in the absence of an initiating substance. These structures are primarily single fibers with uneven fiber distribution, length, and diameter. No spiral or interwoven fibers are observed, making it difficult to form a three-dimensional network. The addition of an initiating substance significantly altered the nanostructure assembled by the self-assembled peptides, with changes in microscopic morphology (Figures 2B-2J). Increased fiber length was observed in all experimental groups, with more fibers appearing and the fibers interwoven into a network. The fine fibers aggregated side by side into ribbon-like fiber bundles, which then interwoven into a three-dimensional structure.

[0279] Experimental Conclusion: The control and experimental groups had the same peptide concentration. Due to the electrostatic interaction between the triggering substance and the peptide molecules, the hydrophilicity of the peptide molecules decreased, resulting in more peptide molecules being observed. The reduction in molecular repulsion promoted self-assembly and high-polymer assembly. Due to the different electron-withdrawing abilities of different triggering substances, the nanostructures of the experimental groups differed. Scanning electron microscopy results demonstrated that a variety of triggering substances can alter the assembly behavior of self-assembled peptides, forming more stable microstructures. The differences in experimental results with different triggering substances indicate that changing the triggering substance can produce hydrogel materials with different structures, facilitating material design and application. Under neutral conditions, the self-assembled peptide solution is mixed with a solution containing the triggering substance to directly form a peptide hydrogel, without the need for an additional complex gelation process. The three-dimensional nanostructure helps simulate the extracellular environment. It can also be carried out in media commonly used in biological and medical research, providing a good physiological environment for three-dimensional cell culture while not changing the chemical composition of the environment, avoiding the introduction of various risks.

[0280] Experiment 2: Transmission Electron Microscopy (TEM)

[0281] The self-assembling peptide is exemplified by SEQ ID NO: 15 (IIIIIGSIIGOGGEGPGGE); the triggering substance is exemplified by polylysine, spermine, gentamicin, lysine, arginine, spermidine, kanamycin, chitosan, and magnesium ions.

[0282] Experimental method: Use ultrapure water to dilute the mother liquor of the self-assembling peptide material obtained in Example 2 (SEQ ID NO: 15) and the three-dimensional mesh scaffold material formed by self-assembly of the self-assembling peptide obtained in Example 3 in response to the triggering substance. In order to obtain the nanoscale morphology of the hydrogel under physiological conditions, incubate at 37°C for 1 hour, take 10 μL and place it on a 300-mesh carbon support film copper grid (Xinxing Bairui), and vacuum dry. Use 2wt.% phosphotungstic acid negative staining solution to stain the sample, each time for 60s, repeat three times. The stained copper grid is placed at room temperature to dry. Use Talos G2200X transmission electron microscope for imaging observation.

[0283] Experimental results: As shown in Figures 3A-3J. Similar to the results of Experiment 1, different initiating substances mixed with self-assembling peptides can all produce the three-dimensional mesh scaffold material of the present invention. The structure formed by the self-assembling material (Figure 3A) is composed of curved and interwoven nanofibers, with uneven fiber distribution, curved fibers, and strong flexibility. The presence of the initiating substance reduces the curvature of the fibers (Figures 3B-3J), significantly increases the fiber length and diameter, and changes the arrangement of the fibers. The fibers are tightly and orderly arranged to form fiber bundles, which are interwoven with each other.

[0284] Experimental Conclusion: Experiment 2 demonstrated that the presence of a triggering substance caused the self-assembling peptides to self-assemble into a gel-like network, which is beneficial for cell adhesion and three-dimensional culture. Combined with Experiments 1 and 2, it was confirmed that the interaction between the self-assembling peptides and the triggering substance altered the self-assembly pathway, prompting them to form a three-dimensional network scaffold material, resulting in a microscopic morphology distinct from that observed without the addition of a triggering substance.

[0285] When the self-assembling peptides used are SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32, or conform to the self-assembling peptide structure described in the present invention, similar experimental results to those of this embodiment can be obtained under the stimulation of the triggering substance, and are not listed in detail one by one.

[0286] Experiment 3: Circular Dichroism (CD)

[0287] The self-assembling peptide is exemplified by SEQ ID NO: 15 (IIIIIGSIIGOGGEGPGGE); the initiating substances are exemplified by polylysine, spermine, magnesium ions, gentamicin, lysine, arginine, and polyarginine.

[0288] Experimental method: Preparation of self-assembling peptide material samples; dilute the self-assembling peptide material mother solution (SEQ ID NO: 15) obtained in Example 2 and the self-assembling peptide obtained in Example 3 that responds to the triggering substance with phosphate buffer solution. In order to obtain a hydrogel structure under physiological conditions that is more in line with the application scenario, all samples were incubated at 37°C for 1 hour before testing. Circular dichroism analysis was performed using a MOS-450 / AF-CD spectrometer (Bio-Logic, Claix, France). The sample was placed in a rectangular quartz cell with an optical path length of 1 mm. The spectral signal with a wavelength of 190 to 260 nm was measured at a resolution of 1 nm and a speed of 0.5 nm / s. After subtracting the solvent background, each sample was scanned three times to obtain the average spectrum.

[0289] Experimental results: Compared to the self-assembled peptide alone, all samples containing the triggering substance exhibited a maximum positive peak near 200nm and a minimum negative peak near 220nm, forming a typical β-pleated secondary structure. Furthermore, the triggering substance shifted the maximum positive peak, affecting the spectral signal intensity to varying degrees, indicating a greater enrichment of β-pleated structure within the material.

[0290] Experimental conclusion: The results of circular dichroism spectroscopy verified the responsiveness of the self-assembling peptide of the present invention to the triggering substance, and explained the mechanism of formation of the peptide hydrogel material of the present invention from the change of the main chain conformation of the self-assembling peptide (Figure 4). The sequence in the relatively hydrophilic sequence segment that is conducive to the formation of the β-turn structure provides the molecular basis for the self-assembling peptide to assemble into a β-folded secondary structure. However, in the absence of a triggering substance, due to the electrostatic repulsion of acidic amino acids, the molecule cannot form a highly ordered β-folded conformation. Experiment 3 shows that the molecular conformation of the self-assembling peptide can be changed by simply mixing the triggering substance and the self-assembling peptide, affecting the composition of its secondary structure and leading to the formation of different nanostructures. Self-assembly systems rich in β-folds often form a large number of hydrogen bonds, which provides a guarantee for the structural stability of the material, and hydrogen bonds, as a highly directional interaction force, are more convenient for regulating the material, further illustrating the adjustability of the peptide hydrogel of the present invention.

[0291] Experiment 4: Rheological properties

[0292] Taking the self-assembled peptide represented by the amino acid sequence SEQ ID NO: 14 (IIIIIGTVIGPGGEGOGGE) as an example, the triggering substances include polylysine, spermine, gentamicin, lysine, magnesium ion, arginine, chitosan, polyarginine, urea, and nicotinamide mononucleotide as examples; the concentrations of the polypeptide and the triggering substance are both 0.5%.

[0293] Experimental method: Dynamic oscillation scanning was performed using the self-loading peptide solution (SEQ ID NO: 14) obtained in Examples 1 and 2 and the self-loading peptide and the initiating substance. The storage modulus (G') and loss modulus (G") were measured using a MARS 60 rheometer on a 20 mm plate. The test temperature was 37°C, consistent with the physiological environment temperature. A 500 μm gap was used, and mineral oil was added to the gap to prevent sample dehydration. Immediately after the preparation of the mixed solution of the self-loading peptide and the initiating substance, it was placed on a plate for a dynamic time scan experiment (DTS) to monitor the changes in the storage modulus (G') and loss modulus (G") over time (1 Hz frequency, 1% strain) for 30 minutes to observe the gel formation rate and gel strength.

[0294] Experimental Results: The storage modulus and loss modulus measured 30 minutes after mixing the self-loading peptide solution with different initiating substances are shown in Figure 5. In all groups with added initiating substances, hydrogels with stable structures and storage moduli greater than 25 Pa were observed within the mixed systems. The mechanical properties of the hydrogels formed by different initiating substances with the same charge concentration ratio as the self-loading peptide were significantly different.

[0295] The peptide hydrogel formation process is shown in the rheological experiment results in Figure 6. When only the self-loading peptide is present at the same peptide concentration, the material is in a solution state, and the lower storage modulus (G') means low viscosity. Its internal molecules can also achieve a low degree of self-assembly, but cannot form a hydrogel material with higher mechanical strength (Figure 6). This is consistent with the previous experimental results. After the peptide solution is mixed with the initiator solution, the storage modulus (G') increases immediately. The initiator can help the self-loading peptide complete gelation within 10 minutes to obtain the peptide hydrogel of the present invention. Within 30 minutes after mixing the peptide solution and the initiator solution, the mechanical strength continues to increase.

[0296] Experimental conclusion: The mechanical strength of the peptide hydrogel of the present invention can effectively provide support and encapsulation effects for cells, drugs, etc., which is conducive to the realization of three-dimensional cell culture, tissue repair, sustained drug release, etc. Experiments have shown that by mixing the peptide solution and the triggering substance solution, a hydrogel material assembled by the self-assembled peptide in response to the triggering substance can be immediately obtained. It is easy to operate and has a short gelation time. This material has multi-responsiveness and can be induced to gel by a variety of triggering substances. By adjusting the type of triggering substance, hydrogel materials with different mechanical strengths can be obtained, and it has adjustability. Different triggering substances have different electron-absorbing abilities, so the peptide hydrogel formed after mixing with the self-assembled peptide has differences in rigidity and structural stability. By utilizing this difference, different triggering substances can be selected as needed to obtain peptide-triggering substance hydrogels with specific mechanical properties, thereby realizing customized design of hydrogel materials.

[0297] The diversity of initiating substances expands the application scenarios of the peptide hydrogel of the present invention, especially some biomedical experiments are particularly sensitive to changes in environmental conditions. The self-assembling peptide can be mixed with different initiating substances to form a hydrogel. When used, the initiating substance with the least impact on the experiment can be selected. It is preferred that the solution environment of the experiment / application itself contains the initiating substance, which greatly improves the convenience of the peptide hydrogel of the present invention and avoids the risks brought about by changes in the environmental conditions and chemical composition of the experiment / application.

[0298] Experiment 5: Determination of shear thinning properties and self-healing ability

[0299] The self-assembling peptide is exemplified by the amino acid sequence SEQ ID NO. 3 (IIIIIGSIIGOGGEGPGGV); the initiating substance is exemplified by polylysine.

[0300] Experimental method: In order to determine whether the hydrogel formed by the self-assembling peptide of the present invention in the presence of an initiating substance has shear thinning properties and self-repair ability, the self-assembling peptide solution and the initiating substance solution are mixed according to a charge concentration ratio of (1-100): (100-1). After stabilization, a single frequency experiment (1 Hz, 1% strain) is performed for half an hour. Then, a shear strain of 300% is applied at a frequency of 1 Hz for 10 seconds to destroy the hydrogel. The recovery of the peptide hydrogel modulus is then measured at a frequency of 1 Hz and 1% strain. This is repeated once to prove that the peptide hydrogel can be destroyed and self-repaired multiple times, with a cycle interval of 10 minutes.

[0301] Experimental results: The peptide hydrogel of the present invention has shear-thinning properties and good self-healing ability. Taking polylysine as an example of the initiating substance, Figure 7 shows that the SEQ ID NO: 13-polylysine hydrogel is destroyed under sufficiently strong strain, and the storage modulus G' and loss modulus G" immediately drop to about 1 Pa. The loss modulus G" is greater than the storage modulus G', indicating that the material is in a liquid state at this time. After the maximum strain is removed, the peptide hydrogel of the present invention can re-gel and the storage modulus G' is restored to more than 90% of the pre-thinning state. Shear strain or shear stress will destroy the gel structure, mainly by breaking the hydrogen bonds inside the material, but this destruction is temporary. Once the strain or stress stops, the hydrogen bonds between the molecules are restored and reassembled, which proves that the material has the ability to self-repair. Moreover, even if the gel is repeatedly destroyed, the material still has the ability to heal itself.

[0302] Experimental conclusion: In practical applications, operations such as pipetting, shaking, injection, filtration, and spraying all impose significant shear strain or shear stress on the material. During these operations, the peptide hydrogel of the present invention thins into a liquid state, and when the operation is terminated, the material reverts to a hydrogel. The shear-thinning properties and self-repair capabilities facilitate the application of the peptide hydrogel material of the present invention in a variety of scenarios. In particular, in tissue repair, the self-assembling peptide solution can be directly injected, where it comes into contact with the initiating substances in the body to assemble into the peptide hydrogel of the present invention.

[0303] Experiment 6: Rheological properties of peptide hydrogel after dilution

[0304] The self-assembling peptide is exemplified by the amino acid sequence SEQ ID NO. 3 (IIIIIGSIIGOGGEGPGGV); and the initiating substance is exemplified by polylysine.

[0305] The experimental method was the same as that of Experiment 4. To simulate the state of the hydrogel during cell separation, a 0.5 wt.% peptide hydrogel was diluted 10-fold, mixed, and immediately placed on a measurement plate. The storage modulus and loss modulus were measured at a frequency of 1 Hz, a strain of 1%, and a temperature of 37°C for 30 minutes. A control group consisted of a sample of the same peptide concentration but without the triggering substance.

[0306] Experimental results: Diluting the peptide hydrogel with a solvent disrupts the material structure, as shown in Figure 8. After ten-fold dilution, the material's storage modulus, G', drops to 1 Pa. Over time, the storage modulus, G', further decreases, falling below the loss modulus, G", demonstrating the characteristics of a solution.

[0307] Experimental Conclusion: The low concentration after dilution prevented the self-assembling peptide from forming a hydrogel, even in the presence of an initiating substance. Therefore, when the peptide hydrogel material of the present invention is used as a cell culture matrix, cell separation and harvesting can be performed simply by dilution followed by centrifugation, which is simple and reduces cell loss.

[0308] in conclusion:

[0309] Experiments 1 through 6 in this Example 4 demonstrate that the self-loading peptide of the present invention can be stimulated by an initiating substance to gel, forming a hydrogel material with sufficient mechanical strength. The gel contains a three-dimensional nanofiber network structure, which is conducive to providing support for cell growth. The self-loading peptide of the present invention is injection-resistant and can form a peptide hydrogel in situ after injection due to its shear-thinning properties and excellent self-repair ability. The diluted peptide hydrogel becomes a solution, which facilitates the separation and harvesting of cells. Experiments 1 through 4 verify that the self-loading peptide hydrogel of the present invention can be obtained by simply mixing the peptide solution with a solution containing an initiating substance. Experiments 1 to 3 illustrate the mechanism of interaction between the self-assembling peptide and the triggering substance. The triggering substance changes the composition of the secondary structure formed by the self-assembling peptide molecules, making the molecular arrangement within the system tend to a more stable structure (Experiment 3); the molecules extend side by side to form a more ordered nanostructure. In Experiments 1 and 2, it is not difficult to find that all the triggering substances change the microscopic morphology of the self-assembling peptide. The peptide molecules assemble to form longer, thicker and more compact nanofibers, and present a network distribution common in gels; Experiment 4 verifies the above analysis. After the triggering substance is mixed with the self-assembling peptide, the storage modulus of the material is significantly improved compared with the peptide solution alone. The supporting capacity of the material is unquestionable, providing a basis for its application in the biomedical field. It is worth noting that in all experiments, all triggering substances can interact with the self-assembling peptide to form the peptide hydrogel of the present invention. Unlike previous hydrogel materials, the gelation conditions of the present invention are not harsh, which expands the application range of the material. In practical applications, suitable triggering substances can be selected as needed to promote the occurrence of gelation. Based on the above experimental results and analysis, the peptide hydrogel of the present invention is simple to prepare, easy to operate, and has a wide range of applications.

[0310] In the above experiments, phosphate buffer, a commonly used solvent in biological, chemical, and medical research, was used solely to illustrate that gelation can be achieved by mixing a self-encapsulated peptide solution with a solution containing an initiator. The present invention's self-encapsulated peptide hydrogels are not limited to phosphate buffer solutions; the solution environment can be flexibly selected based on the application scenario. For example, cell culture media often contain one or more initiating substances. By mixing the self-encapsulated peptide solution with a culture medium containing cells, a cell-encapsulated self-encapsulated peptide hydrogel can be obtained.

[0311] Example 5: Effects of amino acid sequence structure and initiating components on the physical and functional properties of the peptide self-assembly material of the present invention, and verification of the self-assembly mechanism

[0312] Experiment 1: Rheological experiment, the influence of the secondary structure and amino acid sequence of self-assembling peptides

[0313] Experimental method: Preparation of self-assembling peptide material samples: Use the 2 wt% self-assembling peptide solution obtained in Example 2, SEQ ID NO: 1 (IIIIIGSIIGPGGDGPGGV), SEQ ID NO: 2 (IIIIIGSIIGPGGEGPGGV), SEQ ID NO: 3 (IIIIIIGSIIGOGGEGPGGV), SEQ ID NO: 4 (IIIIGSIIGOGGEGPGGV), SEQ ID NO: 5 (IIIIIIGSIIGOGGEGPGGGV), SEQ ID NO: 6 (IIIIIIGSIIGOGGEGPGGV), SEQ ID NO: 7 (IIIIIIGSIIGOGAEGPGGV), SEQ ID NO: 8 (IIIIIIGSIIOGGAEGPGGV), SEQ ID NO: 9 (IIIIIIGSIIGOGGVGPGGV), SEQ ID NO: 10 (IIIIIIGSIIGOGAEGPGGV GPGGV).

[0314] Preparation of hydrogel samples formed by self-assembling peptide solution in response to complete cell culture medium: dilute the 2 wt% self-assembling peptide material stock solution obtained in Example 2 with phosphate buffer, add human tissue fluid, and the final concentration of self-assembling peptide is 0.5%, the final concentration of tissue fluid is 30 v / v%, and the pH is neutral.

[0315] The storage modulus (G') of the mixed solution was measured using a MARS 60 rheometer on a 20 mm plate. To determine the rate of formation of the 3D nanomatrix, the solution was placed on the plate immediately after preparation. A 500 μm gap was used, and mineral oil was added to the gap to prevent sample dehydration, and data collection began. A dynamic time sweep experiment (DTS) was performed to monitor the change in the storage modulus (G') modulus over time (1 Hz frequency, 1% strain) for 2000 seconds, with the modulus value at 900 seconds taken for comparison.

[0316] Experimental results: The mechanical strength of the peptide hydrogel of the present invention can effectively provide support and encapsulation effects for cells, functional molecules, drugs, etc., which is conducive to the realization of three-dimensional cell culture, tissue repair, drug sustained release, etc. Experiments have shown that by mixing the peptide solution and tissue fluid, self-assembling peptides can be immediately obtained to respond to the triggering substance and assemble into a hydrogel material with a storage modulus greater than 10. The operation is convenient and the gelation time is short. Comparing different sequences, it was found (see Figure 9) that SEQ ID NO: 7 (IIIIIGSIIGOGAEGPGGV) > SEQ ID NO: 3 (IIIIIGSIIGOGGEGP GV) > SEQ ID NO: 2 (IIIIIIGSIIGPGGEGPGGV) ≥ SEQ ID NO: 1 (IIIIIIGSIIGPGGDGPG GV); this experimental phenomenon is consistent with the results of Experiment 3, that is, the replacement of glycine (G) with alanine (A) in hydroxyproline and hydrophilic regions can enhance the mechanical strength of the hydrogel formed by the present invention, thereby enhancing the support function of the scaffold material. At the same time, by comparing the results of SEQ ID NO: 5 (IIIIIGSIIGOGGEGPGGGV) and SEQ ID NO: 6 (IIIIIGSIIGOGGEGPGV), it was found that when the number of amino acids forming the β-turn structure was 6 (SEQ ID NO: 5) and 3 (SEQ ID NO: 6), the self-assembling peptide could still form a hydrogel structure with relatively weak elasticity; or when the number of hydrophobic amino acids in the β-turn structure was 3 consecutive (SEQ ID NO: 10) or 4 (SEQ ID NO: 4), the self-assembling peptide could still form a hydrogel structure with relatively weak elasticity.

[0317] In SEQ ID NO:8, we completely destroyed the formation conditions of the first β-turn in SEQ ID NO:7 by adjusting the 10th-11th amino acids GO to OG, forming the sequence SEQ ID NO:8. As a result, we found that SEQ ID NO:8 completely lost its self-assembly response to tissue fluid. This phenomenon further proves that two consecutive β-turns are the most basic conditions for the self-assembly ability of the self-assembling peptides of the present invention. By comparing SEQ ID NO:9 (IIIIIGSIIGOGGVGPGGV) with SEQ ID NO:3 (IIIIIGSIIGOGGEGPGGV), we found that when the end of the β-turn is no longer connected to an acidic amino acid, the aqueous solution of the self-assembling peptide no longer responds to tissue fluid and can form a hydrogel in a neutral aqueous solution. This further verifies that the acidic amino acid attached to the end of the β-turn in the hydrophilic region of at least one self-assembling peptide is a prerequisite for the self-assembling peptide self-assembly material of the present invention to respond to the triggering component.

[0318] Example 6. Properties of the peptide hydrogel material of the present invention obtained in a physiological environment

[0319] Experiment 1: Preparation of the present invention's self-loaded peptide hydrogel under physiological conditions

[0320] The self-assembling peptide is exemplified by SEQ ID NO: 15 (IIIIIGSIIGOGGEGPGGE); the triggering substance is exemplified by human tissue fluid.

[0321] Experimental Method: The self-contained peptide solution (SEQ ID NO: 15) obtained in Example 2 was added to nine volumes of tissue fluid and mixed thoroughly to a final peptide concentration of 0.3 wt%. The solution was allowed to stand for 1 hour, and the sample bottle was inverted. Comparisons were performed with tissue fluid alone, the polar peptide solution alone, and a mixture of the two.

[0322] Experimental results: As shown in Figure 10, sample bottle A contains human tissue fluid, bottle B contains a 0.5 wt.% peptide solution, and bottle C contains a mixture of peptide and tissue fluid. When the sample bottles are inverted, only bottle C forms a gel due to the peptide's response to the triggering substance in the tissue fluid, and the material does not fall out. The addition of the triggering substance will change the state of the peptide solution. Bottles A and B are both liquid. The peptide hydrogel of the present invention can be obtained by the response of the polar peptide to the common in vivo liquid environment (tissue fluid), because tissue fluid contains multiple triggering substances.

[0323] Experimental Conclusion: The peptide hydrogel of the present invention can be obtained by mixing the self-contained peptide solution with tissue fluid. Similar peptide hydrogels can also be obtained by injecting the peptide solution into animals or humans. The peptide hydrogel of the present invention has promising application prospects in the biomedical field. For example, in wound repair, the peptide solution can be directly injected around the wound. The peptide responds to triggering substances such as tissue fluid to form a hydrogel in situ, which helps promote wound healing.

[0324] Experiment 2: Rheological test to detect the formation and mechanical stability of the self-loaded peptide hydrogel of the present invention under physiological environment

[0325] The self-assembling peptides are exemplified by SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32. The triggering substances are exemplified by serum-free culture medium for stem cells (12-725F UltraCULTURE MEDIUM) and human tissue fluid.

[0326] Experimental methods:

[0327] Formation of the peptide hydrogel of the present invention: Dynamic oscillation scanning is performed on a mixture of a self-assembling peptide of amino acid sequence SEQ ID NO: 14 (IIIIIGTVIGPGGEGOGGE) and a serum-free culture medium of stem cells, and a mixture of SEQ ID NO: 11-32 and tissue fluid. The storage modulus (G') and loss modulus (G") of the samples were measured using a MARS 60 rheometer on a 20 mm plate with a 500 μm gap, a temperature of 37°C, a frequency of 1 Hz, and a 1% strain. Mineral oil was added to the gap to prevent sample dehydration. 50 μL of the 5 wt.% self-encapsulated peptide solution (SEQ ID NO: 14) obtained in Example 2 was mixed with 450 μL of serum-free stem cell culture medium and tissue fluid to obtain a mixture with a peptide concentration of 0.5 wt.%. Dynamic time scan (DTS) experiments were immediately performed on the plate to monitor the changes in the storage modulus (G') and loss modulus (G") over time during gel formation. This experiment lasted for 30 minutes to observe the gel formation rate and mechanical strength. For the remaining peptides, the storage modulus (G') and loss modulus (G") values ​​were read 15 minutes after testing.

[0328] Mechanical stability of the peptide hydrogel of the present invention: The self-assembling peptide of SEQ ID NO: 14 was mixed with tissue fluid according to the above method to obtain the peptide hydrogel of the present invention, and then stored at 4°C. The storage modulus (G') and loss modulus (G") were measured using a dynamic rheometer after 1 day, 5 days, 10 days, 20 days, and 30 days.

[0329] Experimental Results: The hydrogel formation process formed by mixing the self-assembling peptide with serum-free stem cell culture medium and tissue fluid was demonstrated using the self-assembling peptide of SEQ ID NO: 14 as an example. Within 30 minutes, a significant increase in G' was observed in the groups containing serum-free stem cell culture medium and tissue fluid, ultimately exceeding 100 Pa, forming a hydrogel (Figures 11A and 11B). This indicates that the addition of the peptide solution to serum-free stem cell culture medium and tissue fluid altered the mechanical properties of the material. The formation of peptide hydrogels in response to triggering substances (serum-free stem cell culture medium and tissue fluid) can be achieved by simply mixing the peptide solution with a system containing the triggering substance. Within 300 seconds after mixing the peptide solution and the triggering substance-serum-free stem cell culture medium and tissue fluid, the material's G' increased rapidly and changed rapidly, then flattened, demonstrating that the peptide of the present invention is rapidly responsive to the triggering substance, even when the triggering substances are multiple substances. Within 15 minutes, the G' of the groups containing both triggering substances tended to flatten, indicating that the rearrangement of the peptide molecules and gelation are essentially complete at this point.

[0330] The self-assembling peptides represented by SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32 of the present invention can respond to gelation with tissue fluid to form peptide hydrogels with certain mechanical strength (Figure 11D). The gel strength obtained by mixing different self-assembling peptides with tissue fluid varies slightly, and the appropriate polar peptide sequence can be selected according to different application scenarios.

[0331] When tissue fluid was used as the triggering agent, the mechanical properties of the peptide hydrogels of the present invention, including the storage modulus and loss modulus, remained essentially stable for up to one month, demonstrating the stability of the peptide hydrogels of the present invention. Although the peptide hydrogels of the present invention are cross-linked by non-covalent bonds, their three-dimensional network structure remained stable for at least 30 days of storage (Figure 11C).

[0332] Experimental Conclusions: Serum-free culture medium for stem cells and tissue fluid can both be used as triggering agents when mixed with the self-assembling peptides to form peptide hydrogels of the present invention. The hydrogels are responsive and possess a certain mechanical strength, capable of supporting 3D cell culture or organoid culture. The self-assembling peptides rapidly react with tissue fluid to achieve gelation, demonstrating the injectable in situ gelation of the peptide hydrogels, which can be used in tissue regeneration and other applications. Furthermore, the peptide hydrogels of the present invention exhibit excellent stability, maintaining stable mechanical properties for at least one month, providing support.

[0333] in conclusion:

[0334] This Example 5 provides a controllable multi-responsive peptide hydrogel of the present invention for application in a physiological environment, thus indicating the possibility of application in vivo. The rapid assembly of peptide hydrogels in response to serum-free culture medium and tissue fluid for stem cells means that the peptide of the present invention can be coated or injected near the wound to form a hydrogel that is beneficial to wound repair. It can also be mixed with tissue fluid to form a hydrogel and then injected into the body to achieve tissue repair, such as cartilage repair. When the peptide hydrogel of the present invention is used, no complicated means are required and no new physical and chemical components are introduced. Common substances for tissue repair such as serum-free culture medium and tissue fluid for stem cells can be used as substances to trigger gelation. The gelation process is rapid, which expands the application range of the peptide hydrogel material of the present invention. The peptide hydrogel of the present invention has good stability, is simple to obtain, and is easy to use and store. It is a good material for cell culture, tissue repair, and sustained drug release.

[0335] Example 7: Application of the peptide hydrogel material of the present invention in the field of biomedicine

[0336] Experiment 1: Supporting effect of the controllable multi-responsive peptide hydrogel of the present invention on red blood cells

[0337] The self-assembling peptides shown in SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32 are taken as examples; the triggering substances are kanamycin, spermine, and arginine as examples; and the cells are red blood cells as an example.

[0338] Experimental method: Anucleated erythrocytes were used as cell models to test the cell support effect of the peptide hydrogel of the present invention. The erythrocyte stock solution was obtained by differential separation, diluted with 1× PBS buffer, and 500 μL of the cell density was added to each centrifuge tube. 8 A red blood cell suspension of 100 μg / mL, 250 μL of the peptide solution of SEQ ID NO: 3 (IIIIIGSIIGOGGEGPGGV) obtained in Example 2, and 250 μL of the triggering substance solution were mixed. The triggering substance in centrifuge tube A was kanamycin, the triggering substance in tube B was spermine, and the triggering substance in tube C was arginine. The charge concentration ratio of the triggering substance to the self-assembling peptide was 1-100:100-1. The final cell density was 1×10 8 cells / mL, with a peptide concentration of 0.5 wt.%. After mixing, the mixture was allowed to stand at 37° C. in a humidified environment with 5% carbon dioxide for 1 hour, the cell sedimentation was observed, and the centrifuge tube was inverted.

[0339] Experimental results: When the centrifuge tubes were turned over, the mixtures in tubes A, B, and C did not fall off, and the red blood cells did not settle and remained evenly distributed (Figure 12).

[0340] Experimental Conclusion: By simply mixing a cell suspension, a peptide solution, and a solution of triggering substances (kanamycin, spermine, and arginine), the peptide hydrogel of the present invention was formed, and a three-dimensional uniform distribution of red blood cells was achieved therein. Combined with the results of Experiment 3 of Example 4, the presence of the triggering substance altered the secondary structure formed by the assembled peptide molecules, leading to the formation of fibers of different morphologies and changes in the distribution of the fibers (Experiments 1 and 2 of Example 4). As a result, the red blood cells were supported by the peptide hydrogel material of the present invention without settling, exhibiting a three-dimensional distribution.

[0341] Experiment 2: Supportive effect of the controllable multi-responsive peptide hydrogel of the present invention on liver cancer cells

[0342] The self-assembling peptide is exemplified by SEQ ID NO: 15 (IIIIIGSIIGOGGEGPGGE); the triggering substances are exemplified by arginine, spermidine, and magnesium ions; and the cells are exemplified by liver cancer cells HepG2.

[0343] Experimental Methods: HepG2 liver cancer cells were cultured in complete high-glucose DMEM (Dulbecco's Modified Eagle Medium) at 37°C in a humidified atmosphere with 5% CO2 (unless otherwise specified, all cell culture conditions were 37°C in a humidified atmosphere with 5% CO2). After digestion, the cells were collected, washed twice with 1×PBS buffer, and resuspended in 1×PBS buffer to obtain 1×10 6 Cells / mL single cell suspension. The cell suspension was mixed with half the volume of Calcein-AM staining solution and incubated at 37°C in the dark for 15 minutes. The stained cells were then inoculated into the peptide hydrogel material of the present invention. The control group was a PBS buffer solution without self-loading peptides, and the experimental group was a mixture of triggering substances and peptides, wherein the triggering substances were arginine, spermidine, and magnesium ions. The cell suspension, triggering substance solution, and peptide solution (SEQ ID NO: 15) were mixed evenly. Cell distribution was observed in a glass-bottom culture dish using a confocal laser scanning microscope. In the above system containing the SEQ ID NO: 15 peptide, the final peptide concentration was 0.3 wt.%, and the charge concentration ratio to the triggering substance was (1-100): (1-100).

[0344] Experimental results: When the system lacked peptides and triggering substances, HepG2 cells naturally settled at the bottom, forming a monolayer (Figure 13A). In the experimental group, the peptide hydrogel material was formed by self-assembly of the peptide in response to the triggering substances through simple mixing, and the cells were added during the gelation process. Therefore, the cells were able to be distributed three-dimensionally within the peptide hydrogel of the present invention, and due to the short gelation time, the cells were evenly distributed (Figures 13B, 13C, 13D).

[0345] Experimental conclusion: The self-assembled peptides in the present invention respond to the triggering substance and assemble into peptide hydrogel materials, forming a structure similar to the extracellular matrix, which has a good supporting effect on liver cancer cells and is a good in vitro 3D cell culture material.

[0346] Experiment 1 and Experiment 2 of this embodiment, through different means of observation, with different cell models and different triggering substances, prove that the peptide hydrogel material of the present invention can effectively support the three-dimensional distribution of cells, simulate the extracellular matrix, affect the extracellular microenvironment, and facilitate the realization of three-dimensional cell culture. And provide a cell inoculation method for the application of the controllable multi-responsive peptide hydrogel of the present invention in three-dimensional cell culture, that is, simply mixing the cell suspension with the peptide solution and the triggering substance solution. It can also be understood that when the system contains cells, the triggering substance and the autonomous peptide, and the charge concentration of the triggering substance and the autonomous peptide is (1-100): (1-100), and the final concentration of the autonomous peptide is ≥0.1wt.%, after mixing, the above-mentioned mixture containing cells is inoculated into a vessel for cell culture to achieve three-dimensional cell culture. The operation is simple, there is no need to adjust the temperature or pH, and no new chemical components will be introduced into the cell culture environment.

[0347] Experiment 3: Verification of the biocompatibility of the controllable multi-responsive peptide hydrogel of the present invention

[0348] The self-assembling peptides are exemplified by SEQ ID NO: 17, SEQ ID NO: 21 and SEQ ID NO: 22; the initiating substance is a cell culture medium, exemplified by high-glucose DMEM complete culture medium; and the cells are exemplified by liver cancer cell HepG2.

[0349] Experimental method: In order to prove that the material of the present invention has good biocompatibility, the CCK-8 (Cell Counting Kit-8) kit was used to detect the activity changes of cells cultured in the material of the present invention for 5 days. 4 HepG2 liver cancer cells were seeded at 100 μL / well in a 96-well plate. The cells were cultured in complete medium containing 0.3 wt.% of SEQ ID NO: 17, SEQ ID NO: 21, and SEQ ID NO: 22, or in complete medium without the self-assembling peptide. After 24, 48, 72, 96, and 120 hours, the culture medium was replaced with fresh complete medium containing 10% CCK-8 solution. The cells were incubated for 30 minutes and the optical density (OD) at 450 nm was measured using a multifunctional enzyme marker. Two to five replicates were performed for each group.

[0350] Experimental results: The activity of HepG2 liver cancer cells cultured in the peptide hydrogel of the present invention was consistently higher than on the first day. Cell activity in all groups containing the self-assembling peptides of the present invention was higher than that in the control group on the fifth day, and cell activity remained unchanged for five days (Figure 14). Furthermore, cells in the groups containing the self-assembling peptides of the present invention did not adhere to the wall, demonstrating that the hydrogel material was formed within the well plate. Cells in all experimental groups maintained good activity.

[0351] Experimental Conclusion: High-glucose DMEM complete medium contains initiators such as spermine, spermidine, arginine, and lysine, which can act as triggers to stimulate the self-assembly of the self-assembled peptides to form the peptide hydrogels described herein. In summary, compared to traditional two-dimensional culture, the peptide hydrogels described herein can promote cell proliferation and maintain cell viability for extended periods of time, exhibiting excellent cell compatibility and enabling three-dimensional cell culture.

[0352] Experiment 4: Application of the Regulated Multi-Responsive Peptide Hydrogel of the Present Invention in Three-Dimensional Cell Culture - Using Muscle Satellite Cells

[0353] The self-assembling peptide is exemplified by SEQ ID NO: 3; the initiating substance is a cell culture medium, exemplified by α-MEM complete culture medium; and porcine muscle satellite cells are used as a cell model to verify that the peptide hydrogel of the present invention can be used as a material for three-dimensional cell culture.

[0354] Experimental method: The two-dimensional cultured muscle satellite cells were digested, counted, and seeded into 48-well plates at a seeding density of 6×10 4 pieces / cm 2 , add the peptide solution obtained in Example 2 to a final volume of 0.5 mL, with final peptide concentrations of 0.1 wt.%, 0.3 wt.%, and 0.5 wt.%, respectively. Mix thoroughly and culture for one week. After 3 days of culture, cells were harvested, digested, and resuspended. The cell suspension was mixed with trypan blue stain for 3 minutes and then counted on a cell counter to determine cell viability. Viability = number of viable cells / total number of cells × 100%. Compare cell morphology under brightfield microscopy after 3 and 7 days of culture, and calculate the diameter of the cell spheres.

[0355] Experimental results: After culturing muscle satellite cells in the peptide hydrogel of the present invention for 3 days, the culture systems containing different polar peptides all maintained good survival rates (Figure 15). The cell survival rates in the groups with 0.1wt.%, 0.3wt.%, and 0.5wt.% of SEQ ID NO: 3 were 93%, 94%, and 91%, respectively.

[0356] While maintaining the vigorous vitality of the cells, the growth morphology of the cells changed (Figures 16A-I). After culturing in the peptide hydrogel of the present invention for 3 days, cell clusters with a diameter of 50-100 μm were observed in the 0.3wt.% and 0.5wt.% systems (Figures 16D, 16G). Muscle satellite cells proliferated massively within 7 days in the peptide hydrogel of the present invention, and more cell clusters and cells were observed on the 7th day (Figures 16B, 16E, 16H). The diameter of the cell spheres in the group containing 0.3wt.% increased to about 80μm (Figure 16F), and the diameter of the cell spheres in the group containing 0.5wt.% increased to about 130μm (Figure 16I).

[0357] Experimental conclusion: The peptide hydrogel of the present invention has good biocompatibility, can keep cells at a high survival rate, and is suitable for three-dimensional cell culture. The self-assembling peptide involved in the present invention can respond to the complete culture medium to form a peptide hydrogel. The peptide hydrogel of the present invention mimics the extracellular matrix, provides three-dimensional growth conditions for cells, and promotes the cell growth state to be closer to that in vivo, forming cell clusters and three-dimensional growth. In this experiment, cell spheres with a diameter of about 100 μm were formed, indicating that the peptide hydrogel of the present invention can be used for the culture of organoids, can ensure the activity of organoids, and can further be used in tissue engineering fields such as organoid transplantation.

[0358] Experiment 5: Application of the Regulatable Multi-Responsive Peptide Hydrogel of the Present Invention in Three-Dimensional Cell Culture - Mesenchymal Stem Cells

[0359] The self-assembling peptide is SEQ ID NO.3 as an example, and the initiating substance is a complete cell culture medium, α-MEM complete culture medium as an example; human umbilical cord-derived mesenchymal stem cells are used as a cell model to verify that the peptide hydrogel material of the present invention can achieve three-dimensional cell culture.

[0360] Experimental method: hMSCs cultured in two dimensions were digested, centrifuged, counted, and resuspended in α-MEM medium containing SEQ ID NO. 3. The cell seeding density was 1×10 6 To more clearly observe cell distribution and morphology, cells were stained with AM / PI live / dead staining after 4 and 7 days of culture, and cell growth was observed using a laser confocal microscope. The peptide hydrogel of the present invention was formed by adding the peptide of SEQ ID NO. 3 to a complete culture medium containing triggering substances such as spermine, arginine, spermidine, and lysine.

[0361] Experimental results: After incubation for 4 days in a mixed system of the self-assembling peptide and complete cell culture medium, mesenchymal stem cells formed obvious cell clusters (Figures 17A and 17C). As the culture time increased, mesenchymal stem cells formed larger and more cell clusters (Figures 17B and 17D), indicating that the cells have good growth and proliferation ability in the peptide hydrogel of the present invention.

[0362] Experimental conclusion: It was verified that the complete cell culture medium, due to the presence of initiating substances such as spermine, spermidine, lysine, arginine, and magnesium ions, can be used as an initiating substance to form a peptide hydrogel with the self-assembling peptide. By simply mixing the polar peptide solution, cell suspension, and cell culture medium, three-dimensional cell culture in the peptide hydrogel of the present invention can be achieved. The preparation method is simple and easy to use. The controllable multi-responsive peptide hydrogel material of the present invention can mimic the microenvironment of natural cell growth, allowing cells to grow close to their natural state. It can be used as a material for three-dimensional cell culture and provides a basis for studying applications such as cell-cell interactions, cell migration, and cell-based drug detection. Stem cell therapy can be achieved by transplanting the peptide hydrogel of the present invention loaded with stem cells into animals or humans.

[0363] Experiment 6: Application of 3D Cell Storage

[0364] Experimental method: This experiment takes the storage of mouse mesenchymal stem cells at 4°C as an example. The isolated mouse mesenchymal stem cells were pipetted evenly and 10 ml of equal volume was dispensed into cryopreservation tubes and pipetted evenly to make the cell concentration between 1×10 6 In the 3D group, mouse serum and 1% double antibody storage solution in 50% MAP + 50% SFEM were added to the peptide SEQ ID NO: 20 (FLIVIGOGIIGOGGEGPGGE) to a final concentration of 0.05%. In the 2D group, mouse serum and 1% double antibody storage solution in 50% MAP + 50% SFEM were added, and then the cells were stored in a 4°C refrigerator.

[0365] Cell viability test:

[0366] (1) Thoroughly mix the stored cells by pipetting, take 500 μL of cell suspension into each 1.5 mL EP tube, add PBS to wash, and centrifuge at 1500 rpm for 10 min;

[0367] (2) Discard the supernatant, resuspend with 150 μL of 1% PBA solution, then add 150 μL of live-dead cell staining working solution, mix thoroughly, incubate at 37°C for 15 min in the dark, and detect using flow cytometry.

[0368] Experimental results: In order to understand the changes in the survival rate of mesenchymal cells during the storage period, we used the Calcein-AM / PI live / dead double staining kit to double-stain them and used a flow cytometer to detect them. As shown in Figure 18, starting from the third day of storage, the survival rate of mouse mesenchymal stem cells in the 3D storage group was significantly higher than that in the 2D storage group, with a significant statistical difference (79% vs. 67%). And on the fifth day of storage, it still had a high cell survival rate (71% vs. 43%), and the survival rate increased by nearly 30%. This shows that the storage effect of the mesenchymal stem cell storage system used in the present invention is better than traditional 2D storage, which greatly improves the cell survival rate.

[0369] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0370] Experiment 7: Application of the hydrogel of the present invention and the cell 3D culture microcarrier in cell proliferation

[0371] Experimental method: The synthesized self-assembling peptide represented by SEQ ID NO: 25 (IIIIIGOGIIGOGGEGPGGV) was dissolved in cell culture medium and vortexed until fully dissolved to obtain a solution with a self-assembling peptide concentration of 0.1%. The solution was then adjusted to pH 7.4 with 0.1M NaOH solution. It was then mixed with polystyrene microcarriers (particle size between 100-500 μm), 10% calf serum was added, and finally, aliquoted to obtain a 1% self-assembling peptide mixed injection containing microcarriers for injection. It can be seen that the solution became opaque and the microspheres were evenly and stably distributed in the self-assembling peptide solution (Figure 19A). The obtained self-assembling peptide mixed injection containing microspheres was placed at room temperature for one month. It can be seen that the obtained injections were all in a uniform suspension state, with no obvious solid-liquid separation. The same experimental method was used to support macroporous gelatin microcarriers (particle size between 100-500 μm) using SEQ ID NO: 19 (IVIVIGSIIGPGGEGOGGV) ( FIG19B ), and to support polylactic acid microspheres (particle size between 100-500 μm) using SEQ ID NO: 26 (Ac-IIIIIIGSIIGPGGEGOGGV) ( FIG19C ).

[0372] This experiment further validated the combined use of SEQ ID NO: 25 (IIIGOGIIGOGGEGPGGV) and polystyrene microspheres for the proliferation of mouse mesenchymal stem cells. Rat bone marrow mesenchymal stem cells (BMSCs) at passage 9 were thawed and cultured in αMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin (Gibco). All cells used in this study were at passages 8-15. Prior to cell seeding, polystyrene microcarriers were immersed in 70% (v / v) ethanol for 1 hour and then exposed to UV light for 30 minutes. Prior to cell seeding, polystyrene microcarriers were incubated in culture medium for 12 hours. BMSCs were seeded and incubated at 37°C in a humidified atmosphere containing 5% CO2. After the cells reached 80% confluence, they were harvested with trypsin containing EDTA. 1-2×10 cells / mg were seeded in a 48-well plate without TC treatment. 4 Cells were seeded at a density of 10 cells / mL with a microcarrier concentration of 1 wt%. A self-assembling peptide scaffold was added on day 2 of culture to achieve 3D culture. Every two days, 80% of the culture medium was withdrawn and replaced with an equal amount of fresh culture medium and self-assembling peptide scaffold. To investigate cell attachment and growth, samples were collected and counted on days 1, 3, 5, 7, and 9 of cell culture. Cells were then analyzed by confocal laser scanning microscopy after staining with an AM / PI live cell staining kit on days 1, 4, and 7.

[0373] Experimental results: First, the support of the self-assembling peptide of the present invention on the polystyrene microcarriers was evaluated under static conditions, and then the proliferation of mouse mesenchymal stem cells in the composite system was verified. Fluorescence microscope images of static culture conditions show (Figure 19D) that a small number of cells are attached to the polystyrene microcarriers on the first day, indicating that the microcarriers have a strong ability to adsorb cells. Subsequently, on the 4th and 7th days of cell culture, visible cell proliferation was shown, and the number of cells on the microspheres increased significantly, indicating that the self-assembling peptide 3D scaffold and microcarrier composite system can maintain cell proliferation and is non-cytotoxic. The cell counting results also support this situation (Figure 19E). After 3 days, the cells in the 2D group showed contact inhibition and no longer proliferated, while the cells in the 3D group remained in a proliferative state, indicating that the 3D culture system is suitable for large-scale culture of stem cells.

[0374] Experiment 8: Application of the hydrogel of the present invention as a dispersed filler for medical aesthetics

[0375] Experimental Method: The synthesized self-assembling peptide represented by SEQ ID NO. 25 (IIIIIGOGIIGOGGEGPGGV) was dissolved in deionized water and vortexed until fully dissolved to obtain a 1.5% self-assembling peptide solution. The pH of the solution was adjusted to 7.4 with 0.1M NaOH solution. Figure 20A shows, from left to right, the self-assembling peptide solution, the precipitated L-polylactic acid microsphere solution, and a mixture of the two. The self-assembling peptide solution was autoclaved and then mixed with L-polylactic acid microspheres (particle size between 25-60 μm). Finally, the solution was aliquoted to obtain a 5% microsphere-containing self-assembling peptide mixed injection for injection. The solution became opaque, and the microspheres were evenly and stably distributed in the self-assembling peptide solution (Figure 20B). The resulting microsphere-containing self-assembling peptide mixed injection was stored at room temperature for one month. The obtained injections were uniformly suspended, with no obvious solid-liquid separation. After adding 1 / 5 tissue fluid, hydrogel formation was observed (Figure 20C).

[0376] Similarly, using the same method and steps, SEQ ID NO.27 (FLIVIGSIIGOGAEGPGGV) at a concentration of 1.5% can also achieve support for 5% polycaprolactone (PCL) microspheres. As shown in Figure 21A, from left to right, there are a transparent SEQ ID NO.27 self-assembling polypeptide solution, a precipitated polycaprolactone (PCL) microsphere solution, and a mixture of the two. The high-temperature sterilized self-assembling peptide solution was mixed with polycaprolactone (PCL) microspheres (particle size between 25-50um) to make the content of polycaprolactone (PCL) microspheres 5%. It can be seen that the microspheres are uniformly and stably distributed in the self-assembling peptide solution (Figure 21B). The obtained self-assembling peptide mixed injection containing microspheres was placed at room temperature for one month. It can be seen that the obtained injections are all in a uniform suspension state, with no obvious solid-liquid separation phenomenon. After adding 1 / 5 tissue fluid, a gel formation phenomenon can be seen (Figure 21C).

[0377] In addition, except for SEQ ID NOs. 25 and 27, the self-assembling peptides represented by SEQ ID NOs. 1-7 and 9-32 of the present invention can have the same function on a variety of other medical aesthetic regeneration microspheres (Table 2).

[0378] Table 2 Common medical aesthetic microspheres:

[0379] Experimental conclusion: This experimental phenomenon proves that the hydrogel material of the present invention, that is, the three-dimensional network scaffold self-assembled by self-assembling peptides in response to tissue fluid, can effectively support medical aesthetic microspheres. It can play a good dispersing and supporting role for the microspheres when not activated. After being activated by tissue fluid, it can quickly form a hydrogel, thereby playing a local shaping function in the tissue; the entire experimental process does not require the addition of other ingredients, and the operation is convenient.

[0380] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0381] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention are intended to be included in the scope of protection of the present invention.

Claims

A broad-spectrum responsive self-assembling peptide comprising a hydrophobic domain and a hydrophilic domain, wherein the hydrophilic domain comprises at least two consecutive β-turn regions capable of forming β-turns. 2 . The broad-spectrum responsive self-assembling peptide according to claim 1 , wherein the at least one β-turn region comprises or is linked to one or more acidic amino acids at the terminal end, preferably comprises or is linked to one acidic amino acid.

3. The broad-spectrum responsive self-assembling peptide according to claim 1, wherein the β-turn region comprises a β-turn motif formed by 3-6 amino acids, and the β-turn motif has the following structure: X1X2X3, X1X2X3X4, X1X2X3X4X5, or X1X2X3X4X5X6, in, X1, X2, X3, X4, X5, and X6 are amino acid residues, and X1, X2, X3, X4, X5, and X6 in each β-turn motif are identical to or different from each other. 4 . The broad-spectrum responsive self-assembling peptide according to claim 3 , which comprises one hydroxyproline (O), preferably the hydroxyproline (O) is contained in the β-turn motif. The broad-spectrum responsive self-assembling peptide according to claim 1 , wherein the hydrophilic domain comprises 2-8 β-turn regions.

6. According to the broad-spectrum responsive self-assembling peptide of claim 3, the β-turn motif in at least one β-turn region contains or is connected to an amino acid selected from glutamic acid (E), valine (V), leucine (L), isoleucine (I), aspartic acid (D) or lysine (K).

7. The broad-spectrum responsive self-assembling peptide according to claim 3, wherein the hydrophilic domain comprises at least one β-turn motif in which X2 is hydroxyproline O, or the hydrophilic domain comprises at least one β-turn motif in which X2 is proline P.

8. The broad-spectrum responsive self-assembling peptide according to claim 3, wherein one or more of X1, X3 and X4 is glycine (G), and / or one or both of X3 and X4 are alanine (A).

9. The broad-spectrum responsive self-assembling peptide according to claim 1, wherein the β-turn motif comprises an amino acid sequence selected from the group consisting of: GPGG (SEQ ID NO.:33), GPGA (SEQ ID NO.:34), GPAG (SEQ ID NO.:35), GPG, GPAA (SEQ ID NO.:36), GPGGG (SEQ ID NO.:37), GOGG (SEQ ID NO.:38), GOGA (SEQ ID NO.:39), GOAG (SEQ ID NO.:40), GOGGA (SEQ ID NO.:37) NO.:41), GOAA (SEQ ID NO.:42), GOG, or GOGV (SEQ ID NO.:43); Preferably, the β-turn motif has an amino acid sequence selected from the group consisting of: GPAGE (SEQ ID NO.:44), GPGGE (SEQ ID NO.:45), GOGAE (SEQ ID NO.:46), GOGGAE (SEQ ID NO.:47), GOGE (SEQ ID NO.:48), GOGGE (SEQ ID NO.:49), GPGAD (SEQ ID NO.:50), GOGGD (SEQ ID NO.:51), GPGGV (SEQ ID NO.:52), GOGGV (SEQ ID NO.:53), GPGGK (SEQ ID NO.:54), GOGGK (SEQ ID NO.:55), GPGAE (SEQ ID NO.:56), GOGAD (SEQ ID NO.:57), GPAAD (SEQ ID NO.:58), GOAAE (SEQ ID NO.:59), GPGGD (SEQ ID NO.:60), GPGGGV (SEQ ID NO.:61), GPGV (SEQ ID NO.:62), GOGGI (SEQ ID NO.:63) or GOGVI (SEQ ID NO.:64).

10. The broad-spectrum responsive self-assembling peptide according to claim 1, wherein the C-terminus of the hydrophilic domain is modified with a reagent or group selected from the following: carboxylic acid, thiol, ketoate, nitrite, phosphonate, thiophosphate, carbonate, sulfate, nitrate, vinyl sulfone, amide, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and / or silane modification.

11. The broad-spectrum responsive self-assembling peptide according to claim 1, wherein the hydrophobic domain comprises 3-10 hydrophobic amino acids. Preferably, the hydrophobic domain comprises 3-7 hydrophobic amino acids, Preferably, the hydrophobic amino acids are selected from the group consisting of isoleucine (I), valine (V), leucine (L), phenylalanine (F) and alanine (A). One or more of .

12. The broad-spectrum responsive self-assembling peptide according to claim 1, wherein the N-terminus of the hydrophobic domain is modified with a reagent or group selected from the group consisting of acetyl, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and / or silane modifications.

13. The broad-spectrum responsive self-assembling peptide according to claim 1, wherein the hydrophobic domain has an amino acid sequence selected from the group consisting of: LLLL (SEQ ID NO.:65), FIIII (SEQ ID NO.:66), IIII (SEQ ID NO.:67), IIII (SEQ ID NO.:68), ILILI (SEQ ID NO.:69), FFLLF (SEQ ID NO.:70), IVIVI (SEQ ID NO.:71), VIVIV (SEQ ID NO.:72), VLFIIV (SEQ ID NO.:72) NO.:73), VLIII (SEQ ID NO.:74), IVALF (SEQ ID NO.:75), LFIVL (SEQ ID NO.:76), FIAIV (SEQ ID NO.:77), FIIIV (SEQ ID NO.:78), Ac-VLFIIV (SEQ ID NO.:79), Ac-IVIVI (SEQ ID NO.:80), Ac-IIIIII (SEQ ID NO.:78) NO.:81), IIIIII (SEQ ID NO.:82), FLIVI (SEQ ID NO.:83), FLIIA (SEQ ID NO.:84), FIFIF (SEQ ID NO.:85), IFIFI (SEQ ID NO.:86), IAILI (SEQ ID NO.:87) or LLLLL (SEQ ID NO.:88).

14. The broad-spectrum responsive self-assembling peptide according to claim 1, further comprising a connecting domain, Preferably, the linker domain comprises 2-8 amino acid residues, preferably 4-5 amino acid residues, Preferably, the linker domain comprises amino acids with small side chains, amino acids with hydroxyl groups on their side chains and / or hydrophobic amino acids away from the hydrophobic region. Preferably, the small side chain amino acid is selected from glycine (G), alanine (A) and serine (S), the amino acid with a hydroxyl group on the side chain is selected from serine (S), threonine (T) and hydroxyproline (O), the hydrophobic amino acid away from the hydrophobic domain is selected from I, V, L, F and A, and the hydrophobic amino acids I, V, F, L, and A are interchangeable. The connecting domain has an amino acid sequence selected from the group consisting of: GSII (SEQ ID NO.: 89), GPOGI (SEQ ID NO.: 90, GPOGV (SEQ ID NO.: 91), GSGII (SEQ ID NO.: 92), GSVI (SEQ ID NO.: 93), GOII (SEQ ID NO.: 94), GPOGL (SEQ ID NO.: 95), OGII (SEQ ID NO.: 96) or GTVI (SEQ ID NO.: 97), wherein, S, T, and O can be interchanged with each other; More preferably, the connecting domain has an amino acid sequence selected from the group consisting of: GSII (SEQ ID NO.:89), GTII (SEQ ID NO.:98), GTVI (SEQ ID NO.:97), GOVI (SEQ ID NO.:99), GSVI (SEQ ID NO.:93), GSVL (SEQ ID NO.:100), GSGII (SEQ ID NO.:92), GSGVI (SEQ ID NO.:101), GOII (SEQ ID NO.:101) NO.:94), OGII (SEQ ID NO.:96), GOGVI (SEQ ID NO.:102) or GOGII (SEQ ID NO.:103). 15 . The broad-spectrum responsive self-assembling peptide according to claim 1 , which has an amino acid sequence selected from the group consisting of the following SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32.

16. A method for forming a scaffold material from the broad-spectrum responsive self-assembling peptide according to any one of claims 1 to 15, the method comprising the steps of initiating the self-assembling peptide to form the scaffold material with a broad-spectrum initiator, Preferably, the method comprises: A self-assembling peptide solution is prepared and added or injected into a system containing at least one initiating substance to form a self-assembling peptide hydrogel under neutral or physiological conditions.

17. The method according to claim 16, wherein the broad-spectrum initiator is a positive charge source substance or a mixed system containing a positive charge source substance, wherein the positive charge source substance comprises a substance with a positively charged group or a positively charged ion; Preferably, the positive charge source material is a biomacromolecule whose number of hydrogen bond acceptors is less than the number of hydrogen bond donors under neutral conditions, a drug, a functional molecule, a metal ion, a microparticle or microsphere with a positively charged surface modification, an animal tissue fluid, and a cell culture system, a cell storage system, or a drug and functional molecule delivery system containing the above components, or a mixed system of one or more thereof. Preferably, the cell culture system is selected from complete cell culture medium, serum-free culture medium, Preferably, the cell storage system is a cell culture storage medium. Preferably, the biomacromolecule is an aminopolysaccharide or a glycoprotein containing an aminopolysaccharide.

18. The method according to claim 16, wherein the drug is a substance having at least one of the following functions: hemostasis, anti-inflammatory, antimicrobial, antifungal, antiviral, antimycoplasma, anticoagulant, analgesic, and promotion of cell, organ, or tissue growth and development. The biochemical molecules containing positive electrochemical groups are preferably selected from basic amino acids, nucleotides, nucleic acids, oligosaccharides, polysaccharides, vitamins, urea, peptides, peptoids, positively charged synthetic polymers, nanoparticles or microparticles, poly(L-lactic acid) microspheres, polycaprolactone microspheres; The positively charged modified synthetic polymer is selected from polylysine and polyarginine; the amino polysaccharide is selected from chitosan; the basic amino acid is selected from lysine and arginine; and the functional molecule is selected from spermine, spermidine, magnesium ion, and nicotinamide mononucleotide.

19. A scaffold material comprising the broad-spectrum responsive self-assembling peptide according to any one of claims 1 to 15, or prepared by the method according to any one of claims 16 to 18; Preferably, the pH of the self-assembling peptide solution is 3.0-11.0, preferably 6.5-8.0; Preferably, the concentration of the self-assembling peptide is 0.1-15 wt.%, preferably 0.3-5 wt.%.

20. The scaffold material according to claim 19, which is a three-dimensional network scaffold material in the form of a hydrogel or a three-dimensional network scaffold material in a dry form.

21. A method for regulating the mechanical properties of the scaffold material according to claim 19 or 20, or regulating the formation time of the scaffold material hydrogel according to claim 19 or 20, said method comprising the steps of adjusting the type, composition and / or concentration of the initiator, Preferably, adjusting the concentration of the initiator is adjusting the relative concentration of the initiator and the self-assembling peptide.

22. The method according to claim 21, further comprising the step of adjusting the concentration of the self-assembling peptide so that the formed hydrogel can significantly disperse and support cells, cell culture microcarriers, and nano- and micro-sized microspheres.

23. A composition comprising the broad-spectrum responsive self-assembling peptide according to any one of claims 1 to 15 and the broad-spectrum initiator according to any one of claims 16 to 18.

24. Use of the broad-spectrum responsive self-assembling peptide of any one of claims 1-15, the method of any one of claims 16-18, the scaffold material of claim 19 or 20, or the composition of claim 23 in one or more of the following: regenerative medicine and tissue regeneration; 2D and 3D cell culture and storage; dispersion and embedding filling of microspheres; dispersion and support of microcarriers in 3D cell culture systems; drug delivery; wound healing; implantable materials; gene therapy; stem cell therapy; and medical cosmetology.

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