WO2024131924A1 - Peptide à auto-assemblage sensible à large spectre et son utilisation - Google Patents

Peptide à auto-assemblage sensible à large spectre et son utilisation Download PDF

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Publication number
WO2024131924A1
WO2024131924A1 PCT/CN2023/140905 CN2023140905W WO2024131924A1 WO 2024131924 A1 WO2024131924 A1 WO 2024131924A1 CN 2023140905 W CN2023140905 W CN 2023140905W WO 2024131924 A1 WO2024131924 A1 WO 2024131924A1
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Prior art keywords
seq
self
peptide
assembling peptide
broad
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PCT/CN2023/140905
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English (en)
Chinese (zh)
Inventor
梁俊
夏亚婷
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矩阵(天津)生物科技有限公司
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Publication of WO2024131924A1 publication Critical patent/WO2024131924A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the invention relates to the field of biomedical materials, in particular to broad-spectrum responsive peptide hydrogel biomaterials.
  • self-assembling peptide hydrogel As a three-dimensional scaffold material, self-assembling peptide hydrogel has good biocompatibility and functional diversity, and is 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 because of their controllable gelation process.
  • stimuli-responsive peptide hydrogels are limited by complex response initiation that is not suitable for application under physiological conditions, or by the endogenous single gelation conditions.
  • the present invention provides a broad-spectrum responsive self-assembling peptide, which can be triggered by a broad-spectrum triggering substance and respond to self-assembly to form a nano-network structure, and a self-assembling peptide solution system that can play a supporting and repairing function in a solution state.
  • 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 ⁇ -turn regions capable of forming ⁇ -turns.
  • the at least one ⁇ -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.
  • At least one ⁇ -turn region comprises an acidic amino acid at a terminal end.
  • the ⁇ -turn region comprises a ⁇ -turn motif formed by 3-6 amino acids, and the ⁇ -turn motif has the following structure:
  • X1, X2, X3, X4, X5, and X6 are amino acid residues, and X1, X2, X3, X4, X5, and X6 in each ⁇ -turn motif are the same as or different from each other.
  • the ⁇ -turn motif comprises one hydroxyproline (O), and preferably, X2 is hydroxyproline (O).
  • the hydrophilic domain comprises 2-8 ⁇ -turn regions.
  • the hydrophilic domain comprises 2, 3, 4, 5, 6, 7, or 8 ⁇ -turn regions.
  • the hydrophilic domain comprises 2-6 ⁇ -turn regions. More preferably, the hydrophilic domain comprises 2-4 ⁇ -turn regions.
  • the hydrophilic domain comprises 2 or 3 ⁇ -turn regions.
  • the ⁇ -turn motif in the at least one ⁇ -turn region comprises or is linked to an acidic amino acid.
  • the ⁇ -turn motif in the at least one ⁇ -turn region comprises or is linked to a glutamic acid (E), valine (V), leucine (L), isoleucine (I), aspartic acid (D) or lysine (K).
  • the hydrophilic domain comprises two ⁇ -turn regions, and the ends of the ⁇ -turn regions comprise acidic amino acids E.
  • the hydrophilic domain comprises two ⁇ -turn regions, wherein one end of the ⁇ -turn region comprises an acidic amino acid E, and the other end of the ⁇ -turn region comprises an amino acid V or K.
  • the hydrophilic domain comprises two ⁇ -turn regions, and the ends of the ⁇ -turn regions comprise an acidic amino acid D.
  • the hydrophilic domain comprises two ⁇ -turn regions, wherein one ⁇ -turn region comprises an acidic amino acid D at one end and the other ⁇ -turn region comprises an amino acid V at one end.
  • the hydrophilic domain comprises at least one ⁇ -turn motif in which X2 is hydroxyproline O, or the hydrophilic domain comprises at least one ⁇ -turn motif in which X2 is proline P.
  • the hydrophilic domain comprises at least one ⁇ -turn motif in which X2 is O.
  • the hydrophilic domain comprises at least one ⁇ -turn motif in which X1 is G.
  • the hydrophilic domain comprises a ⁇ -turn motif in which X2 is O, and a ⁇ -turn motif in which X2 is P.
  • the hydrophilic domain comprises at least one ⁇ -turn motif in which X2 is P.
  • the hydrophilic domain comprises two ⁇ -turn motifs in which X2 is P.
  • one or more of X1, X3 and X4 is glycine (G), and/or one or both of X3 and X4 is alanine (A).
  • the ⁇ -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 I D NO.: 38), GOGA (SEQ ID NO.: 39), GOAG (SEQ ID NO.: 40), GOGGA (SEQ ID NO.: 41), GOAA (SEQ ID NO.: 42), GOG, or GOGV (SEQ ID NO.: 43);
  • the ⁇ -turn motif has an amino acid sequence selected from the group consisting of:
  • X1 and X4 form a hydrogen bond.
  • the hydrophilic domain comprises 2, 3, 4, 5, 6, 7 or 8 ⁇ -turn motifs, preferably, the hydrophilic domain comprises 2 or 3 ⁇ -turn motifs.
  • the beta turn motif has an amino acid sequence selected from the group consisting of:
  • 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).
  • At least one beta turn motif in the hydrophilic domain includes an alanine, thereby improving the mechanical properties of the self-assembling peptide.
  • the C-terminus of the hydrophilic domain can be modified with a reagent or group selected from the group consisting of carboxylic acid, thiol, Ketoate, nitrite, phosphonate, phosphite, carbonate, sulfate, nitrate, vinyl sulfone, amide, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and/or silane modifications.
  • a reagent or group selected from the group consisting of carboxylic acid, thiol, Ketoate, nitrite, phosphonate, phosphite, carbonate, sulfate, nitrate, vinyl sulfone, amide, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyn
  • the amino acid sequence of the hydrophilic domain is more hydrophilic than the amino acid sequence of the hydrophobic domain.
  • the hydrophobic domain comprises 3-10 hydrophobic amino acids.
  • 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.
  • the hydrophobic amino acid is selected from one or more of isoleucine (I), valine (V), leucine (L), phenylalanine (F) and alanine (A).
  • the hydrophobic amino acid is selected from one or more of I, V, L, A, and F.
  • 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.
  • 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.
  • 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), FLFLF (SEQ ID NO.: 70), IVIV I (SEQ ID NO.: 71), VIVIV (SEQ ID NO.: 72), VLFIIV (SEQ ID 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.
  • the amino acid sequence of the hydrophobic domain is hydrophobic compared to the amino acid sequence of the hydrophilic domain.
  • the self-assembling peptide further comprises a connecting domain providing a spacer between the hydrophobic domain and the hydrophilic domain.
  • the connecting domain comprises 2-8 amino acid residues, preferably 4-5 amino acid residues.
  • the connecting domain comprises small side chain amino acids, amino acids with hydroxyl groups on the side chains, and/or hydrophobic amino acids that are far away from the hydrophobic region.
  • the amino acid with a smaller side chain 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 acids away from the hydrophobic domain are selected from I, V, L, F and A, and the hydrophobic amino acids I, V, F, L, 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 are interchangeable with each other;
  • 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), GSG II (SEQ ID NO.: 92), GSGVI (SEQ ID NO.: 101), GOII (SEQ ID NO.: 94), OGII (SEQ ID NO.: 96), GOGVI (SEQ ID NO.: 102) or GOGII (SEQ ID NO.: 103).
  • 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.
  • the self-assembling peptide has a length of 15-50 amino acids, preferably 15-25 amino acids.
  • the self-assembling peptide comprises 2, 3, 4, 5, 6, 7 or 8 ⁇ -turns, preferably, the self-assembling peptide comprises 2 or 3 ⁇ -turns.
  • the self-assembling peptide has an amino acid sequence selected from the following SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32:
  • 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.
  • the three-dimensional mesh scaffold material has a nanostructure.
  • the broad-spectrum initiator is a positive charge source substance or a mixed system comprising a positive charge source substance.
  • the substances include substances with positively charged groups, or positively charged ions.
  • the positive charge source material is a biomacromolecule, drug, functional molecule, animal tissue fluid, and 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.
  • the broad-spectrum triggering substance is selected from physiological molecules containing positive electrochemical groups, biochemical molecules, drugs, functional molecules, metal ions, microparticles or microspheres with positively charged surfaces, or a mixed system of one or more thereof, or an animal tissue fluid containing the aforementioned substances, a complete cell culture medium, a serum-free culture medium, a cell culture storage fluid, or a mixed system of one or more of the drug and functional molecule delivery systems.
  • 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.
  • 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.
  • the polysaccharides and their derivatives include but are not limited to chitin, chitosan, etc.
  • the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.
  • the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, and the like.
  • the antioxidants include but are not limited to niacinamide and other vitamins.
  • the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.
  • the metal ions include, but are not limited to, potassium, calcium, magnesium ions, and the like.
  • the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.
  • the positive charge source is urea or nicotinamide mononucleotide.
  • 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.
  • 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.
  • 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, and tissue growth and development.
  • 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 charged modified synthetic polymers, nanoparticles or microparticles, and cosmetic agents;
  • the positively charged modified synthetic polymers include but are not limited to polylysine and polyarginine;
  • the aminopolysaccharides 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.
  • 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.
  • the initiator is a mixed system of animal tissue fluid.
  • the three-dimensional mesh fiber structure formed by self-assembly can bind at least one of the following substances: biochemical molecules of positive electrochemical groups, drugs, functional molecules, animal tissue fluid, and cell culture storage or drug and functional molecule delivery systems containing the above components.
  • the initiator can be dissolved in a solvent to obtain a solution, and the solution is mixed with the self-assembling peptide solution to obtain a self-assembling peptide-initiator solution with a pH of 6.5 to 10, preferably a pH of 6.5-8.0, more preferably 6.5-7.5, and more preferably 7.0-7.5.
  • 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.
  • 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.
  • the positive charge source material is a biomacromolecule, drug, functional molecule, animal tissue fluid, and 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.
  • the broad-spectrum triggering substance is selected from physiological molecules containing positive electrochemical groups, biochemical molecules, drugs, functional molecules, metal ions, microparticles or microspheres with positively charged surfaces, or a mixed system of one or more thereof, or an animal tissue fluid containing the aforementioned substances, a complete cell culture medium, a serum-free culture medium, a cell culture storage fluid, or a mixed system of one or more of the drug and functional molecule delivery systems.
  • 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.
  • 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.
  • the polysaccharides and their derivatives include but are not limited to chitin, chitosan, etc.
  • the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.
  • the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, and the like.
  • the antioxidants include but are not limited to niacinamide and other vitamins.
  • the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.
  • the metal ions include, but are not limited to, potassium, calcium, magnesium ions, and the like.
  • the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.
  • the positive charge source is urea or nicotinamide mononucleotide.
  • 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.
  • 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.
  • 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, and tissue growth and development.
  • 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-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.
  • 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.
  • the initiator is a mixed system of animal tissue fluid.
  • the three-dimensional mesh fiber structure formed by self-assembly can bind at least one of the following substances: biochemical molecules of positive electrochemical groups, drugs, functional molecules, animal tissue fluid, and cell culture storage or drug and functional molecule delivery systems containing the above components.
  • the initiator can be dissolved in a solvent to obtain a solution, and the solution is mixed with the self-assembling peptide solution to obtain a self-assembling peptide-initiator solution with a pH of 6.5 to 10, preferably a pH of 6.5-8.0, more preferably 6.5-7.5, and more preferably 7.0-7.5.
  • 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.
  • 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.
  • the polypeptides respond and self-assemble into a hydrogel within 120 minutes, preferably within 60 minutes, and more preferably within 10 minutes to form the scaffold material.
  • 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.
  • the broad-spectrum initiating substance and the self-assembling peptide interact with each other to induce the peptide aqueous solution to self-assemble into a scaffold material in the form of a hydrogel.
  • a broad-spectrum initiator substance or a mixed system containing a broad-spectrum initiator substance is added to provide a positive charge, thereby neutralizing the negatively charged acid ions on the self-assembling peptide molecules, thereby reducing the repulsion 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.
  • the three-dimensional mesh scaffold material is in the form of a hydrogel or in a dry form of a hydrogel, such as in the form of a lyophilized powder of a 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 more preferably in the range of 0.2-1 wt.%.
  • the multi-responsive self-assembling peptide of the present invention responds rapidly to the triggering substance, that is, when the triggering substance exists, the multi-responsiveness can self-assemble to form a three-dimensional network scaffold in a relatively short time to achieve gelation.
  • the time of hydrogel formation is affected by the type, composition, and concentration (relative concentration with the self-assembling peptide) of the triggering substance.
  • the self-assembling peptide responds to self-assemble into a hydrogel within 120 minutes, preferably 60 minutes, and more preferably within 10 minutes after contacting the triggering substance.
  • 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.
  • adjusting the concentration of the triggering substance is adjusting the relative concentration of the triggering substance and the self-assembling peptide.
  • the method further comprises the step of adjusting the concentration of the self-assembling peptide.
  • the self-assembly stimulation of the self-assembly peptide by the initiating substance is mainly affected by electrostatic interaction, and the charge concentration ratio of the initiating substance to the self-assembling peptide is 1:100-100:1.
  • the dosage of the self-assembling peptide is as low as possible to achieve the expected effect in order to save material costs. Customization of nanomaterials can be achieved by matching different initiating substances or changing the ratio of initiating substances to self-assembling peptides.
  • the concentration of the self-assembling peptide is preferably above 0.1wt.%, more preferably in the range of 0.15-5wt.%, and more preferably in the range of 0.2-1wt.%.
  • 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.
  • the three-dimensional mesh scaffold material is obtained by the method of the second aspect or the third aspect.
  • the three-dimensional mesh scaffold material is in the form of a hydrogel or in a dry form of a hydrogel, such as in the form of a lyophilized powder of a hydrogel.
  • the three-dimensional mesh scaffold material is in the form of an injectable hydrogel.
  • the three-dimensional mesh scaffold material is a nanostructure.
  • the concentration of the broad-spectrum responsive self-assembling peptide is above 0.1 wt.%, a more preferred concentration range is between 0.15-5 wt.%, and a more preferred concentration range is between 0.2-1 wt.%.
  • the three-dimensional network scaffold material has more ⁇ -folded structures than the self-assembling peptide.
  • the present invention provides a composition comprising the broad-spectrum responsive self-assembling peptide of the first aspect and a broad-spectrum initiator.
  • composition is in the form of being combined together, or in the form of being combined, the latter being that the self-assembling peptide and the broad-spectrum initiator are placed in different containers respectively.
  • 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.
  • the positive charge source material is a biomacromolecule, drug, functional molecule, animal tissue fluid, and 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.
  • the broad-spectrum triggering substance is selected from physiological molecules containing positive electrochemical groups, biochemical molecules, drugs, functional molecules, metal ions, microparticles or microspheres with positively charged surfaces, or a mixed system of one or more thereof, or an animal tissue fluid containing the aforementioned substances, a complete cell culture medium, a serum-free culture medium, a cell culture storage fluid, or a mixed system of one or more of the drug and functional molecule delivery systems.
  • 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.
  • 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.
  • the polysaccharides and their derivatives include but are not limited to chitin, chitosan, etc.
  • the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.
  • the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, and the like.
  • the antioxidants include but are not limited to niacinamide and other vitamins.
  • the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.
  • the metal ions include, but are not limited to, sodium, potassium, calcium, magnesium ions, and the like.
  • the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.
  • the positive charge source is urea or nicotinamide mononucleotide.
  • 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.
  • 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.
  • 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, and tissue growth and development.
  • 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 charged modified synthetic polymers, nanoparticles or microparticles, and cosmetic agents;
  • the positively charged modified synthetic polymers include but are not limited to polylysine and polyarginine;
  • the aminopolysaccharides 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.
  • 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.
  • the initiator is a mixed system of animal tissue fluid.
  • 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 more preferably in the range of 0.2-1 wt.%.
  • the multi-responsive self-assembling peptide of the present invention responds rapidly to the triggering substance, that is, when the triggering substance exists, the multi-responsiveness can self-assemble to form a three-dimensional network scaffold in a relatively short time to achieve gelation.
  • the time of hydrogel formation is affected by the type, composition, and concentration (relative concentration with the self-assembling peptide) of the triggering substance.
  • the self-assembling peptide responds to self-assemble into a hydrogel within 120 minutes, preferably 60 minutes, and more preferably within 10 minutes after contacting the triggering substance.
  • the present invention unexpectedly found that under the conditions of multiple initiators, the three-dimensional mesh scaffold structure formed is more stable, so 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 mesh scaffold material is a nanostructure.
  • the formed hydrogel has a self-repairing function.
  • the gel state of the hydrogel can be destroyed under the action of mechanical force, and then restored to the gel state after the mechanical force disappears.
  • the recovery time does not exceed 10 minutes.
  • the storage modulus of the hydrogel is at least 70% of that before destruction, preferably at least 85% of that before destruction, and more preferably more than 95% of that before destruction.
  • the present invention provides the use 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.
  • 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.
  • the self-assembly to form a gel can be initiated by initiating substances commonly used in the biomedical field, thereby better ensuring the biocompatibility of the hydrogel of the present invention.
  • the broad-spectrum responsive self-assembling peptide provided by the present invention, and the hydrogel prepared therefrom can be used for in vitro three-dimensional culture and storage, to establish a cell model, load cells or organs or organoids, and be injected into an animal or human body for tissue repair; it can also be used as a wound dressing, hemostatic material, etc.; or as a carrier for sustained release of drugs or functional factors; or as a cell preservation material, etc. in biological therapy, tissue engineering, and regenerative medicine.
  • the broad-spectrum responsive self-assembling peptide of the present invention, and the hydrogel prepared therefrom have a wide range of applications and are safe and convenient.
  • the strength of the interaction between different triggering substances and the acidic amino acids of the self-assembling peptide is the basis for the controllability of the 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 a variety of scenarios.
  • the present invention provides a convenient and safe method for preparing controllable broad-spectrum responsive self-assembling peptide hydrogels under physiological conditions, which is suitable for scenes such as laboratories, hospitals, and even the wild, 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 initiating 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.
  • the broad-spectrum responsive self-assembling peptide hydrogel of the present invention can be gelled 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.
  • 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.
  • 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, etc., and has a wide range of applications.
  • 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 directly inject the self-assembling peptide solution of the present invention to form a self-assembling peptide hydrogel with the tissue fluid in the animal/human body, used as a wound dressing, hemostatic material, etc.; or after mixing the drug/functional molecule with the self-assembling peptide solution of the present invention to form a self-assembling peptide hydrogel, it is injected or applied to the wound or
  • Figures 1A-1B are macroscopic images of the self-assembling peptide solution and the mixture of the self-assembling peptide and the initiating substance of the present invention.
  • A is a 0.5wt.% self-assembling peptide solution
  • B is a mixture of the self-assembling peptide and arginine with a peptide concentration of 0.5wt.%
  • Figure 1C shows that with tissue fluid as the initiator, the self-assembling peptide can form a hydrogel, which is still in a gel state after being squeezed out by a syringe.
  • Figures 2A-2J are FESEM images of the self-assembling peptides of the present invention and the mixture of the self-assembling peptides and the initiating substance, and the scale bar is 500nm.
  • Figure 2A is only the self-assembling peptide
  • Figure 2B is a mixture of the self-assembling peptide and polylysine
  • Figure 2C is a mixture of the self-assembling peptide and spermine
  • Figure 2D is a mixture of the self-assembling peptide and gentamicin
  • Figure 2E is a mixture of the self-assembling peptide and lysine
  • Figure 2F is a mixture of the self-assembling peptide and arginine
  • Figure 2G is a mixture of the self-assembling peptide and spermidine
  • Figure 2H is a mixture of the self-assembling peptide and kanamycin
  • Figure 2I is a mixture of the self-assembling peptide and chitosan
  • Figure 2J is a mixture of the self
  • Figures 3A-3J are TEM images of the self-assembling peptides and the mixture of the self-assembling peptides and the initiating substance of the present invention.
  • Figure 3A is only the self-assembling peptide
  • Figure 3B is a mixture of the self-assembling peptide and polylysine
  • Figure 3C is a mixture of the self-assembling peptide and spermine
  • Figure 3D is a mixture of the self-assembling peptide and gentamicin
  • Figure 3E is a mixture of the self-assembling peptide and lysine
  • Figure 3F is a mixture of the self-assembling peptide and arginine
  • Figure 3G is a mixture of the self-assembling peptide and spermidine
  • Figure 3H is a mixture of the self-assembling peptide and kanamycin
  • Figure 3I is a mixture of the self-assembling peptide and chitosan
  • Figure 3J is a mixture of the self-assembling peptide and magnesium ions.
  • FIG. 4 is a circular dichroism spectrum diagram of the self-assembling peptide of the present invention and a mixture of the self-assembling peptide and an initiating substance.
  • FIG. 5 is a comparison of the mechanical properties of the self-assembled peptide of the present invention and a mixture of the self-assembled peptide and different types of initiating substances.
  • 6 is the oscillation time scan results of the self-assembled peptide of the present invention and the mixture of the self-assembled peptide and the triggering substance (polylysine, spermine, gentamicin, lysine, arginine, chitosan, polyarginine, magnesium ion, urea, nicotinamide mononucleotide, respectively).
  • the triggering substance polylysine, spermine, gentamicin, lysine, arginine, chitosan, polyarginine, magnesium ion, urea, nicotinamide mononucleotide, respectively.
  • FIG. 7 shows the results of shear thinning and self-repair experiments of the self-assembled peptide hydrogel of the present invention.
  • FIG8 shows the storage modulus G' and loss modulus G" of the self-loaded peptide hydrogel of the present invention after dilution 10 times.
  • FIG. 9 shows the changes in rheological properties of self-assembling peptides with different sequence structures.
  • Figure 10 is a schematic diagram of the self-assembling peptide of the present invention responding to tissue fluid to form a hydrogel, wherein bottle (A) contains tissue fluid, bottle (B) contains a 0.3wt.% self-assembling peptide solution, and bottle (C) contains a mixture of self-assembling peptide and tissue fluid with a final peptide concentration of 0.3wt.%.
  • Figures 11A-11D are the dynamic rheological test results of the self-assembled peptides of the present invention responding 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 the self-assembled peptides of the present invention responding to serum-free medium for stem cells with a final peptide concentration of 0.5wt.% to form hydrogels
  • Figure 11B shows the self-assembled peptides of the present invention responding to tissue fluid with a final peptide concentration of 0.5wt.%
  • FIG11C shows the stability of the self-loaded peptide hydrogel of the present invention when tissue fluid is used as the triggering substance
  • FIG11D shows the comparison of the modulus of the self-loaded peptide hydrogel formed by the self-loaded peptide of the present invention and the self-loaded peptide hydrogel formed in response to tissue fluid.
  • Figure 12 is a diagram showing the supporting effect of the self-loading peptide hydrogel of the present invention on red blood cells, wherein the triggering substance of tube (A) is kanamycin, the triggering substance of tube (B) is spermine, and the triggering substance of tube (C) is arginine.
  • 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-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.
  • FIG. 14 is the result of cell activity analysis when the self-assembled peptide hydrogel of the present invention is used for three-dimensional cell culture.
  • FIG. 15 shows the cell survival rate of muscle satellite cells after three-dimensional culture in the peptide hydrogel of the present invention for 3 days.
  • 16A-16I are bright field microscope images and cell sphere diameter statistics of muscle satellite cells cultured three-dimensionally in the self-assembled peptide hydrogel of the present invention for 7 days.
  • A is the cells cultured with 0.1wt.% SEQ ID NO: 3 for 3 days
  • B is the cells cultured with 0.1wt.% SEQ ID NO: 3 for 7 days
  • C is the cell sphere diameter distribution diagram of the cells cultured with 0.1wt.% SEQ ID NO: 3 within 7 days
  • D is the cells cultured with 0.3wt.% SEQ ID NO: 3 for 3 days
  • E is the cells cultured with 0.3wt.% SEQ ID NO: 3 for 7 days
  • F is the cell sphere diameter distribution diagram of the cells cultured with 0.3wt.% SEQ ID NO: 3 within 7 days
  • G is the cells cultured with 0.5wt.% SEQ ID NO: 3 for 3 days
  • H is the cells cultured with 0.5wt.% SEQ ID NO: 3 for 7 days
  • 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).
  • FIG. 18 shows the effect of not using (2D) and using (3D) the self-assembling peptide scaffold solution on the cell survival of mouse mesenchymal stem cells during refrigerated storage.
  • Figures 19A-19E show the suspension support effect of self-assembling peptides on microcarriers, and the use of this system for the proliferation and culture of mouse mesenchymal stem cells, where 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 microscope image under static culture conditions, and E is a cell counting result.
  • Figures 20A-20C show that the self-assembling peptide solution can stably disperse the L-polylactic acid microspheres.
  • A shows that the microspheres precipitate in the aqueous solution, but are evenly suspended and dispersed after mixing with the self-assembling peptide solution;
  • B shows that the self-assembling peptide mixture of the microspheres is liquid;
  • C shows that after the self-assembling peptide mixture of the microspheres obtained in B is mixed with tissue fluid, the self-assembling peptide/L-polylactic acid microsphere solution quickly forms a hydrogel.
  • Figures 21A-21C show that the self-assembling peptide solution can stably disperse polycaprolactone.
  • A shows that the microspheres precipitate in the aqueous solution, but are evenly suspended and dispersed after mixing with the self-assembling peptide solution;
  • B shows that the self-assembling peptide mixture of the microspheres is liquid;
  • C shows that after the self-assembling peptide mixture of the microspheres obtained in B is mixed with tissue fluid, the self-assembling peptide/L-polylactic acid microsphere solution quickly forms a hydrogel.
  • the present invention obtains a self-assembling peptide by ingeniously 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 matching the hydrophobic amino acids and the hydrophilic amino acids.
  • 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 a hydrogel is a hydrogel material having a three-dimensional network scaffold structure.
  • the broad-spectrum self-assembling peptides of the present invention are in a neutral liquid state during use under human physiological conditions, thereby avoiding the risks caused by adjusting pH or introducing other exogenous substances, such as certain metal salt ions, specific proteins, and the like.
  • the broad-spectrum self-assembling peptide of the present invention has a stronger supporting effect due to the presence of hydroxyproline (O) in the ⁇ -turn motif of the hydrophilic domain.
  • the inventors found that the presence of hydroxyproline (O) compared to proline (P) in the ⁇ -turn motif of the hydrophilic domain resulted in a stronger supporting effect for the self-assembling peptide.
  • the acidic amino acids of the self-assembling peptide of the present invention interact with the initiator to induce the peptide aqueous solution to self-assemble into a peptide hydrogel.
  • the addition of the initiator can give a positively charged substance, so that the negatively charged acid radical ions on the polypeptide molecules are neutralized, thereby reducing the repulsion between the polypeptide molecules, and the polypeptide molecules then realize the self-assembly of the polypeptide molecules through hydrophobic interaction and hydrogen bonds, and finally form a nano three-dimensional network structure.
  • the present invention provides an adjustable broad-spectrum responsive self-assembling peptide hydrogel and a preparation and application method thereof.
  • the adjustable broad-spectrum responsive self-assembling peptide hydrogel contains a triggering 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.
  • the tissue fluid itself can be used as a triggering 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.
  • the broad-spectrum responsive self-assembling peptide hydrogel of the present invention has a broad-spectrum initiation, which is reflected in the fact that it can 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 the illusion of experimental results. Some 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.
  • 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 technicians to learn complicated operating procedures, and does not introduce any exogenous substances to bring unnecessary risks.
  • the presence of the triggering substance under physiological conditions 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 hydrogel storage modulus is not less than 20Pa.
  • 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 an adjustable broad-spectrum responsive self-assembling peptide hydrogel.
  • 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, and the personalized design of nanomaterials can be realized. For example, according to the optimal growth environment of different cell lines, self-assembling peptide hydrogels with different supporting capacities are obtained.
  • 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 re-form the hydrogel. The storage modulus will be restored to at least 60% before the gel is destroyed, and the hydrogel self-repair time 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, filtering, etc.
  • the polypeptide hydrogel still has the ability to self-repair after suffering multiple damages.
  • the self-assembling peptide hydrogel of the present invention is provided in an injectable form and can be gelled in situ after injection.
  • 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.
  • the self-assembling peptides involved in the present invention may be a mixture of one or more self-assembling peptides.
  • the self-assembling peptides include a relatively hydrophobic hydrophobic domain and a relatively hydrophilic hydrophilic domain, wherein the relatively hydrophilic hydrophilic domain contains at least two continuous ⁇ -turn motifs forming a ⁇ -turn structure, wherein at least one ⁇ -turn motif terminal is 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.
  • 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 be A neutral or alkaline solution is used as a dissolving agent, and after dissolution, the pH of the polypeptide solution is adjusted to 6.5 to 10.0, preferably 7.0-7.5.
  • the dissolving agent includes sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonia water, and a mixture thereof, which 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.
  • the controllable multi-responsive self-assembling peptide hydrogel of the present invention is formed by the self-assembling peptide responding to and assembling 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 there are only a small number of large protein molecules in the tissue fluid), 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.
  • the drug has at least one of the following functions: hemostasis, anti-inflammatory, antimicrobial, antifungal, antiviral, anti-mycoplasma, anticoagulation, analgesia, and promotion of cell, organ, and tissue growth and development.
  • the drug 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 aminopolysaccharides 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, sperm
  • the initiating substance comprises a mixture of a plurality of the above-mentioned substances.
  • the initiating substance is a mixture of the above-mentioned initiating substances, such as cell culture medium and animal tissue fluid.
  • the three-dimensional mesh fiber structure formed by self-assembly can bind at least one of the following substances: biochemical molecules of positive electrochemical groups, drugs, functional molecules, animal tissue fluid, and cell culture storage or drug and functional molecule delivery systems containing the above components.
  • the initiating substance can be dissolved to obtain a solution, and the solution is mixed with the self-assembling peptide solution to obtain a self-assembling peptide-initiating substance solution with a pH of 6.5 to 10, preferably a pH of 6.5-8.0, more preferably 6.5-7.5, and more preferably 7.0-7.5.
  • triggering substances are selected to illustrate the technical effects of the self-assembling peptide hydrogels of the present invention.
  • 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 verification data on the multi-responsive characteristics and functions of the self-assembling peptides of the present invention. It is understandable that other triggering substances for which detailed data are not provided in the present invention also have equivalent or similar effects.
  • the self-assembling peptide hydrogel of the present invention is formed by self-assembling peptides in response to triggering substances under neutral conditions. The principle is briefly described below:
  • the side chains of the self-assembling peptides of the present invention carry negative charges, and the electrostatic interaction between the initiating substance and the self-assembling peptides can neutralize part or all of the negative charges in the self-assembling peptide solution system, reduce the electrostatic repulsion between molecules, and promote the occurrence of self-assembly.
  • the self-assembling peptides of the present invention cannot be independently assembled into hydrogels when there is a lack of initiating substances 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 tight molecular arrangement.
  • the sequence segment of the ⁇ -turn structure makes the acidic amino group more active, which further plays a role.
  • the peptide molecules tend to establish hydrogen bonds with water molecules, increasing the difficulty of the self-assembling peptide to self-assemble into a three-dimensional mesh scaffold structure.
  • the carbonyl group on the self-assembling peptide will produce an electrostatic attraction with the initiating substance.
  • the negative charges on the self-assembling peptide molecules are partially or completely "shielded" and are electrically neutral, the electrostatic repulsion between molecules is reduced, and the hydrophobic interaction and hydrogen bonds cause the molecules to tend to be orderly aggregated and regularly distributed.
  • 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, affects the hydrophilicity of the self-assembling peptides, and makes The solubility of the material decreases, forming a hydrogel structure.
  • the initiating substance affects the self-assembly process of the self-assembling peptide to form a highly ordered aggregate dominated by a ⁇ -folded structure, forming a stable fiber structure with strong orientation.
  • 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 macro 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 is as low as possible to achieve the desired effect in order to save material costs. Customization of nanomaterials can be achieved by matching different initiating substances or changing the ratio of initiating substances to self-assembling peptides.
  • the concentration of the self-assembling peptide is preferably above 0.1wt.%, more preferably in the range of 0.15-5wt.%, and more preferably in the range of 0.2-1wt.%.
  • the multi-responsive self-assembling peptide of the present invention responds rapidly to the triggering substance, that is, when the triggering substance exists, the multi-responsiveness can self-assemble to form a three-dimensional network scaffold in a relatively short time to achieve gelation.
  • the time of hydrogel formation is affected by the type, composition, and concentration (relative concentration with the self-assembling peptide) of the triggering substance.
  • the self-assembling peptide responds to self-assemble into a hydrogel within 120 minutes, preferably 60 minutes, and more preferably within 10 minutes after contacting the triggering substance.
  • the preparation method of the controllable multi-responsive self-assembling peptide hydrogel of the present invention is simple and easy to operate.
  • the self-assembling peptide solution is mixed with the initiating substance solution or the self-assembling peptide solution is added to the environment containing the initiating substance to obtain the self-assembling peptide hydrogel material.
  • 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 substance, which 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 realize 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 transferred to a culture dish, a culture bottle, a cell culture well plate and other culture equipment to realize three-dimensional cell culture.
  • the culture conditions are consistent with traditional two-dimensional cell culture.
  • the cell culture medium contains initiating substances such as spermine, lysine, arginine, spermidine, magnesium ions, etc., it can stimulate the self-assembling peptide 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.
  • the self-assembling peptide hydrogel material of the present invention is also suitable as a hemostatic composition or to promote wound healing. It can be injected or applied to the wound site.
  • the tissue fluid in the patient's body serves as a triggering substance, which can cause the self-assembling peptides to respond to form peptide hydrogel materials at the injection site or wound site, thereby playing a supporting role or promoting healing.
  • the peptide or protein hydrogels currently used and widely studied for cell culture need to consider the triggering mechanism of the hydrogel when applying the materials.
  • the mechanical properties of the self-assembling peptide hydrogel of the present invention can be adjusted by only changing the type, composition, and concentration (relative concentration with the self-assembling peptide) of the triggering substance.
  • Hydrogel is a hydrophilic polymer material that can form a three-dimensional network structure by 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 innate biological functions and must undergo important post-processing to induce the desired response in vivo.
  • 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), polyacryl
  • Hydrogels have the following properties:
  • the polymer contains a large number of hydrophilic groups, which can absorb dozens of times more water than itself, and has the characteristics of swelling but not dissolving in water, and has good water retention capacity;
  • Biodegradability Some natural polymer materials are biodegradable, which can avoid secondary damage caused by implant removal. It is these unique advantages that make hydrogels shine in biomedical materials.
  • a hydrogel is obtained by encapsulating water.
  • Peptide Self-Assembly is a short chain of amino acids with polar domains. When dissolved in neutral solvents and physiological salt concentrations, 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-thrombotic, degradable and easily metabolized.
  • nanofibers have the same size scale as natural ECM fibers, can be easily designed to mimic the stiffness of various soft tissues, and can 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.
  • Self-assembling peptides can be used as hydrogels for wound treatment as drug carriers, and can also be used as self-assembling peptide nanofibers for cancer treatment to continuously release small molecules, growth factors, and monoclonal antibodies.
  • self-assembling peptides can be used to promote angiogenesis in regenerated tissues and to repair skin wounds using self-assembling peptide-based scaffolds.
  • the field of injectable therapeutic hydrogels for self-assembling peptides is still in its infancy.
  • the alternating hydrophilic and hydrophobic amino acid residues in the self-assembling peptides enable the self-assembling peptides to retain a large amount of water to form a hydrogel.
  • the side chains of the hydrophilic residues can directly interact with water, and the water molecules form inclusion complexes to surround the side chains of the hydrophobic residues.
  • the number of hydrophobic residues and hydrophilic residues in the self-assembling peptides requires clever design and proportioning. If there are too many hydrophobic residues, the self-assembling peptides 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 peptides are highly water-soluble and therefore cannot 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.
  • 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 may be named using either a three-letter or a one-letter code, e.g., alanine (Ala, A); arginine (Arg, R); asparagine (Asp, N); aspartic acid (D), cysteine (C); glutamine (Q); glutamic acid (E); glycine (Gly, G); histidine (His, H), isoleucine (Ile, I); leucine (Leu, L), 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); selenocy
  • a "peptide” is an amino acid chain.
  • a peptide is 2 to 40 amino acids in length.
  • self-assembly refers to the aggregation of self-assembling peptides under normal environmental conditions to form an ordered structure.
  • ⁇ -fold refers to a relatively extended periodic folded zigzag main chain conformation in a polypeptide chain, arranged in parallel or antiparallel. Thus forming a ⁇ -fold (sheet).
  • 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).
  • Parallel arrangement can include peptide translation causing the two ends of the peptide to be staggered with each other. At least half of the length of the peptide is involved in the interaction between peptides.
  • antiparallel arrangement the polypeptides are usually arranged in a line to provide two flush ends. This is a typical end-to-end complementary peptide.
  • ⁇ -turn is an irregular secondary structure in proteins that causes a change in the direction of the polypeptide chain. ⁇ -turns often occur at the corner where the peptide chain makes a 180° turn. ⁇ -turns are composed of 3-5 amino acid residues, with the second residue being proline (P) or hydroxyproline (O).
  • the ⁇ -turn motif generally refers to a turn structure stabilized by hydrogen bonding between the carbonyl oxygen atom of the nth amino acid residue and the amide proton of the n+3th amino acid residue.
  • ⁇ -turns are also called ⁇ -elbows, reverse elbows or ⁇ -loops, and are used to connect ⁇ -chains.
  • hydrophilcity used in the present invention refers to a property of being inclined to repel water or being completely insoluble in water.
  • hydrophilicity in the present invention refers to the property of easily absorbing water and having a strong polar group that easily interacts with water.
  • Hydrophilic amino acids also known as polar amino acids, have polar R groups that can generally form hydrogen bonds with water molecules, so they have a certain affinity for water molecules. Hydrophilic amino acids include: S, T, Y, C, U, N, Q, D, E, O, R, K, H.
  • Hydrophobic amino acids also known as non-polar amino acids, have non-polar R groups and have low or very low 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.
  • Nanostructure refers to a structure with nanometer size. Nanostructures can be any shape in one, two, or three dimensions, including nanofilms, nanofibers, nanorods, nanowires, nanofiber networks, nanospheres, nanohelices, and mixtures thereof.
  • the surface of a nanostructure has a one-dimensional structure at the nanoscale, i.e., the surface thickness of the object is between 0.1nm and 100nm.
  • Nanotubes have two dimensions of nanometer size, with a diameter of 0.1 to 100nm and a length that may be larger.
  • Spherical nanoparticles have three dimensions in nanometer size, i.e., the size of the particle nanoparticle in each spatial dimension is 0.1 to 100 nanometers.
  • 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 it is tested 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.
  • response and “initiate” are used to indicate that the self-assembling peptide forms a hydrogel in response to different initiators, or that different initiators are used to induce the self-assembling peptide to form a hydrogel, and the two have the same meaning.
  • initiator and initiating substance are used interchangeably, and the two have the same meaning.
  • 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 table SEQ ID NO.1-32.
  • the self-assembling peptide concentration is adjusted using the mother solution to obtain a self-assembling peptide material of a predetermined concentration. For example, a certain amount of the self-assembling peptide mother solution is diluted with PBS buffer to obtain a 0.5wt.% self-assembling peptide solution.
  • 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), so as to obtain a self-assembling, three-dimensional network scaffold controllable self-assembling peptide hydrogel material with multiple initiating substances, and 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).
  • 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, 0.08wt.% lysine solution, arginine solution, gentamicin solution (calculated based on the presence of 4 amino groups), kanamycin solution, urea solution, nicotinamide mononucleotide solution, 0.06wt.% 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 1wt.% 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.
  • polylysine, chitosan, and polyarginine are taken as examples.
  • a mixed solution is prepared according to a mass fraction ratio of macromolecules to self-assembling peptides of 1:1 (ensuring that the charge concentration is (1-100): (1-100)).
  • the solvent is phosphate buffer, and the pH value is neutral
  • the self-assembling peptide solution with a concentration of 1wt.% 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 concentration of the self-assembling peptide in the final solution is 0.5wt.%.
  • the mixed system of the initiator substance and the self-assembling peptide solution is generally prepared according to the desired final self-assembling peptide concentration. Taking 1wt.% of the self-assembling peptide solution and mixing it with an equal volume of the initiator substance can obtain the peptide hydrogel of the present invention with a self-assembling peptide concentration of 0.5wt.%.
  • the initiator substance can be a surface positively charged gelatin porous microsphere, tissue fluid, complete cell culture medium, serum-free culture medium (animal-free culture medium, animal protein-free culture medium, chemically defined culture medium).
  • the triggering substance is arginine as an example
  • the 0.5wt.% peptide solution (see Figure 1A) is in liquid state. After the sample bottle is inverted, the solution flows back and concentrates at the mouth of the bottle.
  • the peptide concentration is 0.5wt.% of the peptide hydrogel that self-assembles in response to the triggering substance arginine (see Figure 1B). The sample bottle is inverted, and the material will not fall due to the formation of hydrogel.
  • the self-assembling peptide can form a hydrogel, which is still in a gel state after being squeezed out by a syringe (see Figure 1C). It also has the same or similar effects on peptide hydrogel materials formed in response to other types of triggering substances.
  • a 2% concentration of the self-assembling peptide quickly forms a hydrogel after being mixed with an equal volume of human tissue fluid.
  • the hydrogel can be sucked up with a syringe and remains in a gel state after injection and extrusion. It also has the same or similar effects on peptide hydrogel materials formed in response to other types of triggering substances.
  • the self-assembling peptide is taken as an example of the self-assembling peptide shown by the amino acid sequence SEQ ID NO: 14 (IIIIIGTVIGPGGEGOGGE); the initiating substance is taken as an example of polylysine, spermine, gentamicin, lysine, arginine, spermidine, kanamycin, chitosan, and magnesium ions.
  • the self-assembled peptides of the present invention can assemble into amorphous fibrous nanostructures (Figure 2A) in the absence of initiating substances under neutral conditions.
  • the fibers are mainly single fibers, with uneven fiber distribution, length and diameter. No spiral fibers or interwoven fibers are observed, and it is difficult to form a three-dimensional network.
  • the addition of initiating substances significantly changes the nanostructure assembled by the self-assembled peptides, and the microscopic morphology changes (Figures 2B-2J).
  • an increase in fiber length was observed, and more fibers appeared, and the fibers were interwoven into a network.
  • the fine fibers gathered side by side into ribbon-like fiber bundles, and the fiber bundles were interwoven into a three-dimensional structure.
  • the control group and the experimental group have the same peptide concentration. Due to the electrostatic interaction between the triggering substance and the peptide molecules, the hydrophilicity of the peptide molecules decreases, so more peptide molecules are observed, and the reduction of molecular mutual repulsion promotes self-assembly and high-polymer assembly. Due to the different electron-absorbing abilities of different triggering substances, there are differences in the nanostructures of the experimental groups. The results of scanning electron microscopy can prove that a variety of triggering substances can change the assembly behavior of self-assembled peptides and form a more stable microstructure.
  • the self-assembled peptide is exemplified by SEQ ID NO: 15 (IIIIIGSIIGOGGEGPGGE); the initiating substance is exemplified by polylysine, spermine, gentamicin, lysine, arginine, spermidine, kanamycin, chitosan, and magnesium ions.
  • Experiment 2 proves that the presence of the triggering substance makes the self-assembling peptide self-assemble into a gel-like network distribution that is conducive to cell adhesion and three-dimensional culture. Combining Experiments 1 and 2, it can be determined that the interaction between the self-assembling peptide and the triggering substance changes the path of the self-assembly of the self-assembling peptide, prompting the self-assembling peptide to form a three-dimensional network scaffold material, forming a microscopic morphology that is completely different from that without the addition of the triggering substance.
  • the self-assembling peptide is exemplified by SEQ ID NO: 15 (IIIIIGSIIGOGGEGPGGE); the initiating substance is exemplified by polylysine, spermine, magnesium ion, gentamicin, lysine, arginine, and polyarginine.
  • Experiment 3 illustrates 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, resulting in the formation of different nanostructures.
  • Self-assembly systems rich in ⁇ -folds often form a large number of hydrogen bonds, which provide 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, which further illustrates the adjustability of the peptide hydrogel of the present invention.
  • the triggering substance is 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%.
  • the self-loading peptide solution (SEQ ID NO: 14) obtained in Examples 1 and 2 and the self-loading peptide and the initiating substance were used for dynamic oscillation scanning.
  • the storage modulus (G’) and loss modulus (G”) were measured on a 20 mm plate using a MARS 60 rheometer.
  • the test temperature was 37°C, consistent with the physiological environment temperature.
  • a gap of 500 ⁇ m was used, and mineral oil was added to the gap to prevent sample dehydration.
  • DTS dynamic time scanning experiment
  • the peptide hydrogel formation process is shown in the rheological experiment results in Figure 6.
  • a 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.
  • the storage modulus (G') increases immediately. The initiator can help the self-assembled peptide to 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.
  • the mechanical strength of the peptide hydrogel of the present invention can effectively provide support and encapsulation effects for cells and drugs, which is conducive to achieving three dimensional cell culture, tissue repair, drug sustained release, etc.
  • a hydrogel material assembled by the self-assembled peptide in response to the triggering substance can be immediately obtained.
  • the operation is convenient and the gelation time is short.
  • This material has multi-responsiveness and can be induced to gel by a variety of triggering substances.
  • hydrogel materials with different mechanical strengths can be obtained, which is adjustable.
  • triggering substances have different electron-absorbing abilities, so the rigidity and structural stability of the peptide hydrogel formed after mixing with the self-assembled peptide are different. 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.
  • the diversity of triggering 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-contained peptide mixed with different triggering substances can form a hydrogel.
  • the triggering 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 triggering substance, which greatly improves the convenience of the peptide hydrogel of the present invention and avoids the risks caused by changes in the environmental conditions and chemical composition of the experiment/application.
  • the self-assembling peptide is taken as an example of the self-assembling peptide shown by the amino acid sequence SEQ ID NO.3 (IIIIIGSIIGOGGEGPGGV); the initiating substance is taken as an example of polylysine.
  • the peptide hydrogel of the present invention has shear-thinning properties and good self-healing ability.
  • Figure 7 shows that 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 1Pa.
  • the loss modulus G” is greater than the storage modulus G’, indicating that the material is in a liquid state at this time.
  • the peptide hydrogel of the present invention can re-gel and the storage modulus G’ is restored to more than 90% before thinning.
  • 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 acting, the hydrogen bonds between the molecules are restored and reassembled, which proves that the material has the ability to self-repair. Moreover, the material still has the ability to self-heal even if the gel is repeatedly destroyed.
  • the self-assembling peptide is taken as an example of the self-assembling peptide shown by the amino acid sequence SEQ ID NO.3 (IIIIIGSIIGOGGEGPGGV); the initiating substance is taken as an example of polylysine.
  • the experimental method was the same as that of Experiment 4. To simulate the state of the hydrogel during cell separation, the 0.5wt.% peptide hydrogel was diluted 10 times, mixed and immediately placed on the measurement plate, and the storage modulus and loss modulus were tested at a frequency of 1 Hz, a strain of 1%, and a temperature of 37°C for 30 min.
  • the control group was a sample of the same peptide concentration without the triggering substance.
  • the peptide hydrogel of the present invention was diluted with a solvent to destroy the material structure, as shown in Figure 8. After dilution ten times, the storage modulus G' of the material dropped to 1Pa. As time went by, the storage modulus G' further decreased and was lower than the loss modulus G", which showed the characteristics of a solution.
  • the self-loading peptide of the present invention can be stimulated by the triggering substance to gel, forming a hydrogel material with a certain mechanical strength, and 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 supports injection and can form a peptide hydrogel in situ after injection because it has shear thinning properties and excellent self-repairing ability; the diluted peptide hydrogel becomes a solution, which is convenient for separating and harvesting cells.
  • Experiments 1 to 4 verify that to obtain the self-loading peptide hydrogel of the present invention, it is only necessary to directly mix the peptide solution with the solution containing the triggering substance.
  • Experiments 1 to 3 illustrate the mechanism of interaction between self-assembled peptides and initiating substances.
  • the initiating substances change the composition of the secondary structure formed by the self-assembled peptide molecules, so that the molecular arrangement in the system tends to a more stable structure (experiment 3); the molecules extend side by side to form a more ordered nanostructure.
  • the peptide molecules assemble to form longer, thicker and tighter nanofibers, and present a common mesh distribution in the gel;
  • Experiment 4 verifies the above analysis.
  • the storage modulus of the material is significantly improved compared with the single peptide solution.
  • the supporting capacity of the material is beyond doubt, which provides a basis for its application in the biomedical field. It is worth noting that in all experiments, all initiating substances can interact with the self-assembled peptides 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 initiating 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.
  • phosphate buffer which is commonly used in the fields of biology, chemistry, and medicine research, was selected as the solvent. It is only used to illustrate that gelation can be achieved by mixing the self-loading peptide solution substance in the solution containing the trigger.
  • the self-loading peptide hydrogel of the present invention is not limited to being obtained in phosphate buffer, and the solution environment can be flexibly selected according to the application scenario.
  • the cell culture medium often contains one or more triggering substances.
  • 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
  • Preparation of hydrogel samples formed by self-assembling peptide solution in response to complete cell culture medium dilute the 2wt% self-assembling peptide material mother solution obtained in Example 2 with phosphate buffer, add human tissue fluid, the final concentration of self-assembling peptide is 0.5%, the final concentration of tissue fluid is 30v/v%, and the pH is neutral.
  • 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 tested on a plate immediately after preparation. A 500 ⁇ m gap was used, and mineral oil was added to the gap to prevent sample dehydration, and data collection began. A dynamic time scan experiment (DTS) was performed to monitor the change in the storage modulus (G’) modulus over time (1 Hz frequency, 1% strain) for 2000 seconds, and the modulus value at 900 seconds was taken for comparison.
  • DTS dynamic time scan experiment
  • 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.
  • the self-assembling peptide can be immediately assembled into a hydrogel material with a storage modulus greater than 10 in response to the triggering substance. The operation is convenient and the gelation time is short.
  • SEQ ID NO: 8 we completely destroyed the formation conditions of the first ⁇ -turn in SEQ ID NO: 7 by adjusting the 10th-11th amino acid GO in SEQ ID NO: 7 to OG, forming the sequence SEQ ID NO: 8; as a result, we found that SEQ ID NO: 8 completely lost the self-assembly response to tissue fluid. This phenomenon further proves that two consecutive ⁇ -turns are the most basic conditions for the self-assembly peptide in the present invention to have the ability to self-assemble.
  • Example 6 Properties of the peptide hydrogel material of the present invention obtained in a physiological environment
  • the self-assembling peptide is taken as SEQ ID NO: 15 (IIIIIGSIIGOGGEGPGGE) and the initiating substance is taken as human tissue fluid.
  • the self-contained peptide solution (SEQ ID NO: 15) obtained in Example 2 was added to nine times the volume of tissue fluid, mixed evenly, and the final peptide concentration was 0.3wt.%, left to stand for 1h, and the sample bottle was turned over. Comparison was made with only tissue fluid, only the polar peptide solution, and a mixture of the two.
  • sample bottle A contains human tissue fluid
  • B contains 0.5wt.% peptide solution
  • C contains a mixture of peptide and tissue fluid.
  • sample bottles are inverted, only bottle C forms gel due to the peptide's response to the triggering substance tissue fluid, and the material does not fall off.
  • the addition of the triggering substance will change the state of the peptide solution, and 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 the tissue fluid contains a variety of triggering substances.
  • the peptide hydrogel of the present invention can be obtained by mixing the self-loading peptide solution with tissue fluid, and similar peptide hydrogels can be obtained by injecting the peptide solution into animals or humans.
  • the peptide hydrogel of the present invention has good application prospects in the field of biomedicine.
  • the peptide solution when used for wound repair, can be directly injected around the wound, and the peptide responds to the triggering substance such as tissue fluid to form a hydrogel in situ, which is beneficial to promote wound healing.
  • the self-assembled peptides are exemplified by SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32: the initiating substances are exemplified by serum-free culture medium for stem cells (12-725F UltraCULTURE MEDIUM) and human tissue fluid.
  • 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 medium for stem cells, and a mixture of SEQ ID NO: 11-32 and tissue fluid.
  • the storage modulus (G') and loss modulus (G") of the sample were measured on a 20 mm plate with a gap of 500 ⁇ m and a temperature of 37°C at a frequency of 1 Hz and a strain of 1%. To prevent the sample from dehydrating, mineral oil was added to the gap.
  • Example 2 50 ⁇ L of the 5 wt.% self-loading peptide solution (SEQ ID NO: 14) obtained in Example 2 was mixed with 450 ⁇ L of serum-free medium for stem cells and tissue fluid to obtain a mixture with a peptide concentration of 0.5 wt.%. The mixture was immediately placed on a plate for a dynamic time scan experiment (DTS) to monitor the changes in the storage modulus (G') and loss modulus (G") of the gel formation process over time. The experiment lasted for 30 minutes to observe the formation rate and mechanical strength of the gel; for the remaining polypeptides, the storage modulus (G') and loss modulus (G") were read 15 minutes after the test. Energy modulus (G') and loss modulus (G") modulus values.
  • DTS dynamic time scan experiment
  • 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 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 of storage.
  • the peptide hydrogel formed by the response of the peptide of the present invention to the triggering substance can be obtained by simply mixing the peptide solution with a system containing the triggering substance.
  • the G’ of the material increased rapidly, changed rapidly, and then the area was flat, indicating that the responsiveness of the peptide of the present invention to the triggering substance is rapid, even if the triggering substance is a variety of substances.
  • the G’ of the groups containing the two triggering substances tended to be flat, indicating that the rearrangement of the peptide molecules has been basically completed at this time, and the gelation has been completed.
  • the self-assembling peptides shown in 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.
  • the mechanical properties of the peptide hydrogel of the present invention remain substantially stable for up to one month, reflecting the stability of the peptide hydrogel of the present invention.
  • the peptide hydrogel of the present invention is cross-linked by non-covalent bonds, its three-dimensional network structure can still be stably stored for at least 30 days ( Figure 11C).
  • Serum-free culture medium and tissue fluid for stem cells can be used as initiating substances to mix with the self-assembling peptide to form the peptide hydrogel of the present invention, which is sensitive to response and has a certain mechanical strength, and can provide support for 3D cell culture or organoid culture.
  • the self-assembling peptide can quickly respond to tissue fluid to achieve gelation, proving that the peptide hydrogel of the present invention can be injected to achieve in situ gelation and is used in tissue regeneration and other aspects.
  • the peptide hydrogel of the present invention has good stability and can maintain stable mechanical properties for at least one month, providing a supporting effect.
  • This Example 5 provides the application of the controllable multi-responsive peptide hydrogel of the present invention under physiological conditions, thus indicating that it is possible to be applied in vivo.
  • the rapid assembly of peptide hydrogels in response to serum-free culture medium and tissue fluid for stem cells means that the peptides 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.
  • tissue repair such as serum-free culture medium and tissue fluid for stem cells can be used as substances that 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 easy to obtain, and is easy to use and store. It is a good material for cell culture, tissue repair, and drug sustained release.
  • Example 7 Application of the peptide hydrogel material of the present invention in the field of biomedicine
  • the self-assembled 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; and the cells are red blood cells.
  • the erythrocyte stock solution was obtained by differential separation and diluted with 1 ⁇ PBS buffer.
  • 500 ⁇ L of erythrocyte suspension with a cell density of 2 ⁇ 10 8 /mL, 250 ⁇ L of SEQ ID NO: 3 (IIIIIGSIIGOGGEGPGGV) peptide solution obtained in Example 2, and 250 ⁇ L of initiator solution were added to each centrifuge tube and mixed well.
  • the initiator in centrifuge tube A was kanamycin
  • the initiator in tube B was spermine
  • the initiator in tube C was arginine.
  • the initiator and the self-assembled peptide had a charge concentration of 1.5 wt %.
  • the final cell density was 1 ⁇ 10 8 cells/mL and the peptide concentration was 0.5 wt.%.
  • the mixture was placed in a humidified environment at 37°C and 5% carbon dioxide for 1 hour, the cell sedimentation state was observed and the centrifuge tube was inverted.
  • the self-assembled peptide is exemplified by SEQ ID NO: 15 (IIIIIGSIIGOGGEGPGGE); the initiating substances are arginine, spermidine, and magnesium ions; and the cells are exemplified by liver cancer cells HepG2.
  • HepG2 liver cancer cells were cultured in a complete medium, high-glucose DMEM (Dulbecco's Modified Eagle Medium), at 37°C and 5% CO2 in a humid environment (unless otherwise specified, all cell culture conditions were 37°C and 5% carbon dioxide in a humid environment). After digestion, the cells were collected, washed twice with 1 ⁇ PBS buffer, and resuspended with 1 ⁇ PBS buffer to obtain a 1 ⁇ 10 6 cell/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 in the peptide hydrogel material of the present invention, the control group was a PBS buffer 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, respectively.
  • the cell suspension, triggering substance solution, and peptide solution (SEQ ID NO: 15) were mixed evenly.
  • the cell distribution was observed in a glass-bottomed culture dish using a confocal laser scanning microscope.
  • the final peptide concentration was 0.3 wt.%, and the charge concentration ratio to the initiating substance was (1-100):(1-100).
  • the self-assembled peptides in the present invention respond to the triggering substances 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.
  • Experiment 1 and Experiment 2 of this embodiment use different observation methods, different cell models and different triggering substances to 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.
  • a cell inoculation method is provided for the application of the adjustable multi-responsive peptide hydrogel of the present invention in three-dimensional cell culture, that is, a simple mixing of cell suspension with peptide solution and triggering substance solution.
  • the system contains cells, triggering substances 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.%
  • the above-mentioned mixture containing cells is inoculated into a vessel for cell culture to realize 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.
  • the self-assembled peptides are SEQ ID NO: 17, SEQ ID NO: 21 and SEQ ID NO: 22 as examples;
  • the initiating substance is cell culture medium, taking high-glucose DMEM complete culture medium as an example;
  • the cells are liver cancer cells HepG2 as an example.
  • 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.
  • HepG2 liver cancer cells with a cell density of 5 ⁇ 10 4 /mL were inoculated into a 96-well plate, and the volume of each well was 100 ⁇ L.
  • the cells were cultured in complete culture medium containing 0.3wt.% of SEQ ID NO: 17, SEQ ID NO: 21 and SEQ ID NO: 22 and complete culture medium without adding self-assembling peptides.
  • the culture medium containing 10% CCK-8 was replaced.
  • the solution was added with fresh complete medium, incubated for 30 minutes, and the optical absorption value (OD) at 450 nm was measured with a multifunctional enzyme marker. All groups were set up in 2-5 parallel groups.
  • liver cancer cells HepG2 cultured in the peptide hydrogel of the present invention was always higher than that on the first day.
  • the cell activity on the fifth day in all groups containing the self-assembling peptides of the present invention was higher than that in the control group, and the cell activity remained unchanged for five days ( Figure 14).
  • the cells in the groups containing the self-assembling peptides of the present invention did not grow attached to the wall, proving that hydrogel materials were generated in the well plate.
  • the cells in all experimental groups maintained good activity.
  • the high-glucose DMEM complete medium contains initiating substances such as spermine, spermidine, arginine, and lysine, which can be used as an initiating substance to stimulate the self-assembly of the self-assembled peptides to form the peptide hydrogel of the present invention.
  • initiating substances such as spermine, spermidine, arginine, and lysine, which can be used as an initiating substance to stimulate the self-assembly of the self-assembled peptides to form the peptide hydrogel of the present invention.
  • the peptide hydrogel of the present invention can promote cell proliferation and maintain cell viability for a longer period of time, has good cell compatibility, and can achieve three-dimensional cell culture.
  • the self-assembling peptide is taken as SEQ ID NO: 3 as an example; the initiating substance is a cell culture medium, taking ⁇ -MEM complete culture medium as an example; 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.
  • 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-loading 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.
  • the surface 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.
  • the self-assembling peptide takes SEQ ID NO.3 as an example, and the initiating substance is a complete cell culture medium, taking ⁇ -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.
  • hMSCs cells cultured in two dimensions were digested, centrifuged, counted, and resuspended in a complete medium ⁇ -MEM medium containing SEQ ID NO.3.
  • the cell seeding density was 1 ⁇ 10 6 cells/mL, and the final concentration of the peptide was 0.1wt.%.
  • the cells were stained with AM/PI live and dead cells after 4 and 7 days of culture, and the growth state of the cells was observed using a laser confocal microscope.
  • the peptide SEQ ID NO.3 was added to a complete medium containing initiating substances such as spermine, arginine, spermidine, and lysine to form the peptide hydrogel of the present invention.
  • peptide hydrogel of the present invention loaded with stem cells into an animal or human body can achieve stem cell therapy.
  • This experiment takes the 4°C storage of mouse mesenchymal stem cells as an example.
  • the separated mouse mesenchymal stem cells were blown evenly, and 10 ml of equal amount was dispensed into cryopreservation tubes, and blown evenly to make the cell concentration about 1 ⁇ 10 6 cells/mL.
  • mouse serum and 1% double antibody 50% MAP+50% SFEM storage solution were added, and the polypeptide SEQ ID NO: 20 (FLIVIGOGIIGOGGEGPGGE) was added to a final concentration of 0.05%; mouse serum and 1% double antibody 50% MAP+50% SFEM storage solution were added to the 2D group, and then stored in a 4°C refrigerator;
  • 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, and there is no obvious solid-liquid separation phenomenon.
  • the same experimental method was used to support macroporous gelatin microcarriers (particle size between 100-500 ⁇ m) using SEQ ID NO: 19 (IVIVIGSIIGPGGEGOGGV) ( Figure 19B), and to support polylactic acid microspheres (particle size between 100-500 ⁇ m) using SEQ ID NO: 26 (Ac-IIIIIIGSIIGPGGEGOGGV) ( Figure 19C).
  • SEQ ID NO: 25 (IIIIIGOGIIGOGGEGPGGV) and polystyrene microspheres for the proliferation of mouse mesenchymal stem cells.
  • the 9th generation rat bone marrow mesenchymal stem cells (BMSCs) were thawed and cultured in ⁇ MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco). All cells used in this study were at passages 8-15. Prior to cell inoculation, the polystyrene microcarriers were immersed in 70% (v/v) ethanol for 1 hour and then exposed to ultraviolet light for 30 minutes.
  • the polystyrene microcarriers Prior to cell inoculation, the polystyrene microcarriers were immersed in 70% (v/v) ethanol for 1 hour and then exposed to ultraviolet light for 30 minutes. The carriers were incubated in the culture medium for 12 h. BMSCs were seeded and incubated at 37 °C in a humidified environment with 5% CO 2. After the cells reached 80% confluence, they were harvested with trypsin containing EDTA. Cells were seeded at a density of 1-2 ⁇ 10 4 cells per mg in 48-well plates without TC treatment, with a microcarrier concentration of 1 wt%, and self-assembling peptide scaffolds were added on the second day of culture to achieve 3D culture.
  • SEQ ID NO.27 FLIVIGSIIGOGAEGPGGV
  • PCL polycaprolactone
  • Figure 21A from left to right are the transparent SEQ ID NO.27 self-assembling peptide solution, the precipitated polycaprolactone (PCL) microsphere solution, and the 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%.

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Abstract

La présente invention concerne un peptide à auto-assemblage sensible à large spectre, un hydrogel formé par le peptide à auto-assemblage, et une utilisation de l'hydrogel.
PCT/CN2023/140905 2022-12-23 2023-12-22 Peptide à auto-assemblage sensible à large spectre et son utilisation WO2024131924A1 (fr)

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* Cited by examiner, † Cited by third party
Title
MAKABE KOKI, MCELHENY DAN, TERESHKO VALENTIA, HILYARD AARON, GAWLAK GRZEGORZ, YAN SHUDE, KOIDE AKIKO, KOIDE SHOHEI: "Atomic structures of peptide self-assembly mimics", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 103, no. 47, 21 November 2006 (2006-11-21), pages 17753 - 17758, XP093185189, ISSN: 0027-8424, DOI: 10.1073/pnas.0606690103 *

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