Liquid positive charge-responsive self-assembling peptides and applications thereof

Abstract

The present invention relates to a liquid, positively charged, responsive self- assembling peptide, a liquid scaffold material formed from the self-assembling peptide, and applications of the liquid scaffold material.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and more specifically to liquid positive charge responsive peptide biomaterials. Background Technology

[0002] Self-assembling peptides have attracted much attention due to their excellent biocompatibility and diverse functions. Studies have found that self-assembling peptides can spontaneously form aggregates with certain structures under specific responses, thereby exerting functions and characteristics such as cell support, antibacterial activity, and tissue repair. They exhibit properties and advantages that differ from monomeric self-assembling peptide molecules, and therefore have promising application prospects in the field of biomedical materials.

[0003] Several self-assembling peptides have been reported, capable of responding to various factors such as temperature, pH, light, metal ions, and specific proteins, thereby undergoing self-assembly to form network scaffold structures and performing functions such as drug delivery, drug screening and evaluation, tissue engineering, and 3D cell culture. However, these response conditions may limit practical applications and increase application risks. For example, high or low pH values ​​and high salt ion concentrations can damage cells; introducing exogenous initiating substances may pose certain human safety risks in clinical applications; and the setting of self-assembly initiation conditions, such as temperature and light, becomes a necessary consideration during application, thus limiting the application conditions of self-assembling peptides. Furthermore, currently reported self-assembling peptide-based scaffold systems must be applied at high concentrations in a gel state, leading to challenges such as difficulty in dispersing and separating cells or functional molecules, difficulties in injection or coating applications, inconvenience in stirring and adding nutrients during cell culture, and high application costs.

[0004] Therefore, developing a self-assembled peptide solution system that can respond to and self-assemble into a nano-network structure under physiological conditions, cell storage and culture environments, and substances widely present in the human body, and can perform support and repair functions in solution, has great application significance. Summary of the Invention

[0005] This invention provides a liquid positive charge responsive self-assembling peptide, which can be initiated and responded to by a positive charge source substance to form a nano-network structure, and can perform support and repair functions in solution.

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

[0007] In some embodiments, the at least one β-turn region contains or is linked to one or more acidic amino acids at its end, preferably containing or being linked to one acidic amino acid.

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

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

[0010] X1X2X3, X1X2X3X4, X1X2X3X4X5, or X1X2X3X4X5X6

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

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

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

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

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

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

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

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

[0019] In some embodiments, the hydrophilic domain includes two β-turn regions, the ends of which contain the acidic amino acid E.

[0020] In some embodiments, the hydrophilic domain includes two β-turn regions, one of which terminates with an acidic amino acid E, and the other terminates with an amino acid V or K.

[0021] In some embodiments, the hydrophilic domain includes two β-turn regions, the ends of which contain the acidic amino acid D.

[0022] In some embodiments, the hydrophilic domain includes two β-turn regions, one of which terminates with an acidic amino acid D, and the other terminates with an amino acid V.

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

[0024] In some embodiments, the hydrophilic domain contains at least one β-turn motif with X2 = 0.

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

[0026] In some embodiments, the hydrophilic domain includes a β-turn motif with X2 of 0 and a β-turn motif with X2 of P.

[0027] In some embodiments, the hydrophilic domain contains at least one β-turn motif with X2 as P.

[0028] In some embodiments, the hydrophilic domain contains two β-turn motifs with X2 as P.

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

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

[0031] GPGG (SEQ ID NO:35), GPGA (SEQ ID NO:36), GPAG (SEQ ID NO:37), GPG, GPAA (SEQ ID NO:38), GPGGG (SEQ ID NO:39), GOGG (SEQ ID NO:40), GOGA (SEQ ID NO:41), GOAG (SEQ ID NO:42), GOGGA (SEQ ID NO:42) NO:43), GOAA (SEQ ID NO:44), GOG, or GOGV (SEQ ID NO:45).

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

[0033] GPAGE (SEQ ID NO:46), GPGGE (SEQ ID NO:47), GOGAE (SEQ ID NO:48), GOGGAE (SEQ ID NO:49), GOGE (SEQ ID NO:50), GOGGE (SEQ ID NO:51), GPGAD (SEQ ID NO:52), GOGGD (SEQ ID NO:53), GPGGV (SEQ ID NO:54), GOGGV (SEQ ID NO:55), GPGGK (SEQ ID NO:56), GOGGK (SEQ ID NO:57), GPGAE (SEQ ID NO:58), GOGAD (SEQ ID NO:59), GPAAD (SEQ ID NO:60), GOAAE (SEQ ID NO:61), GPGGD (SEQ ID NO:62), GPGGGV (SEQ ID NO:63), GPGV (SEQ ID NO:64), GOGGI (SEQ ID NO:65) or GOGVI (SEQ ID NO:66).

[0034] In some implementations, X1 and X4 form hydrogen bonds.

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

[0036] In some embodiments, the β-turn motif has an amino acid sequence selected from:

[0037] GOGG (SEQ ID NO:40), GPGG (SEQ ID NO:35), GOGA (SEQ ID NO:41), GOAG (SEQ ID NO:42), GPGA (SEQ ID NO:36) or GPAG (SEQ ID NO:37).

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

[0039] In some embodiments, the C-terminus of the hydrophilic domain may be modified with a reagent or group selected from: carboxylic acids, thiols, ketoates, nitrites, phosphonates, thiophosphates, carbonates, sulfates, nitrates, vinyl sulfones, amides, alcohols, aldehydes, amines, imines, maleimides, thiols, vinyl sulfones, azides, alkynes, alkenes, esters, thioesters, aryl groups, and / or silanes.

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

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

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

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

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

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

[0046] LLLL (SEQ ID NO:67), FIIII (SEQ ID NO:68), IIII (SEQ ID NO:69), IIII (SEQ ID NO:70), ILILI (SEQ ID NO:71), FLFLF (SEQ ID NO:72), IVIVI (SEQ ID NO:73), VIVIV (SEQ ID NO:74), VLFIIV (SEQ ID NO:74) NO:75), VLIII (SEQ ID NO:76), IVALF (SEQ ID NO:77), LFIVL (SEQ ID NO:78), FIAIV (SEQ ID NO:79), FIIIV (SEQ ID NO:80), Ac-VLFIIV (SEQ ID NO:81), Ac-IVIVI (SEQ ID NO:82), Ac-IIIIII (SEQ ID NO:82) NO:83), IIIIII (SEQ ID NO:84), FLIVI (SEQ ID NO:85), FLIIA (SEQ ID NO:86), FIFIF (SEQ ID NO:87), IFIFI (SEQ ID NO:88), IAILI (SEQ ID NO:89) or LLLLL (SEQ ID NO:90).

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

[0048] In some embodiments, the self-assembling peptide further includes a linker domain that provides a spacer region between the hydrophobic and hydrophilic domains.

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

[0050] In some embodiments, the linker domain comprises small side-chain amino acids, amino acids with hydroxyl groups on their side chains, and / or hydrophobic amino acids located away from the hydrophobic region.

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

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

[0053] In some embodiments, the hydrophobic amino acid far from the hydrophobic domain is selected from I, V, L, F, and A, and the hydrophobic amino acids I, V, F, L, and A can be interchanged.

[0054] In some embodiments, the linker domain has an amino acid sequence selected from the following: GPOGI (SEQ ID NO:91), GPOGV (SEQ ID NO:92), GPOGL (SEQ ID NO:93), GSII (SEQ ID NO:94), GSGII (SEQ ID NO:95), GSVI (SEQ ID NO:96), GOII (SEQ ID NO:97), OGII (SEQ ID NO:98), or GTVI (SEQ ID NO:99), wherein S, T, and O are interchangeable.

[0055] More preferably, the linker domain has an amino acid sequence selected from:

[0056] GSVL (SEQ ID NO:100), GSII (SEQ ID NO:101), GTII (SEQ ID NO:102), GTVI (SEQ ID NO:103), GOVI (SEQ ID NO:104), GSVI (SEQ ID NO:105), GSGII (SEQ ID NO:106), GSGVI (SEQ ID NO:107), GOII (SEQ ID NO:108), OGII (SEQ ID NO:109), GOGVI (SEQ ID NO:110) or GOGII (SEQ ID NO:111).

[0057] In some embodiments, one or more Gs are further included between the hydrophobic domain and the linker domain to enhance the softness and flexibility of the self-assembled peptide.

[0058] In some embodiments, the length of the self-assembled peptide is 15-50 amino acids, preferably 15-25 amino acids.

[0059] In some embodiments, the self-assembled peptide contains 2, 3, 4, 5, 6, 7 or 8 β turns, preferably, the self-assembled peptide contains 2 or 3 β turns.

[0060] In some embodiments, the self-assembled peptide has an amino acid sequence selected from SEQ ID NOs:1-7 and SEQ ID NOs:9-34:

[0061] IIIIIIGSIIGPGGDGPGGV (SEQ ID NO.: 1),

[0062] IIIIIGSIIGPGGEGPGGV (SEQ ID NO.: 2),

[0063] IIIIGSIIGOGGEGPGGV(SEQ ID NO.:3)、

[0064] IIIIGSIIGOGGEGPGGV(SEQ ID NO.:4)、

[0065] IIIIIGSIIGOGGEGPGGGV(SEQ ID NO.:5)、

[0066] IIIIIGSIIGOGGEGPGV(SEQ ID NO.:6)、

[0067] IIIIIGIIGOGAEGPGGV(SEQ ID NO:7)、

[0068] IIIIIGSIIOGGAEGPGGV(SEQ ID NO:8)、

[0069] IIIIIGSIIGOGGVGPGGV(SEQ ID NO.:9)、

[0070] IIIIIIGSIGOGAEGPGGVGPGGV(SEQ ID NO.:10)

[0071] FLIVIGSIIGOGAEGPGGV(SEQ ID NO.:11)、

[0072] FLIIAGSIIGPGGDGOGGV(SEQ ID NO.:12)

[0073] IIIIIGOGIIGPGGDGPGGV(SEQ ID NO.:13)、

[0074] IIIIIGTVIGPGGEGOGGE(SEQ ID NO.:14)、

[0075] IIIIIGTVIGPGGEGOGGK(SEQ ID NO.:15)、

[0076] IAILIGTVIGPGGEGOGGE(SEQ ID NO.:16)、

[0077] IIIIGTVIGPGGEGOGGV(SEQ ID NO.:17)、)

[0078] IIIIIGOGIIGPGGEGPGGV(SEQ ID NO.:18)、

[0079] IIIIIGOGIIGPGGDGPGGD(SEQ ID NO.:19)、

[0080] FLIVIGOGIIGOGGEGPGGV(SEQ ID NO.:20)

[0081] IVIVIGSGIIGPGGDGPGGV(SEQ ID NO:21)、

[0082] IVIVIGOGIIGOGGDGOGGV(SEQ ID NO.:22)、

[0083] IIIIIGSIIGPGGEGOGGV(SEQ ID NO.:23)、

[0084] FIIIVGSIIGPGGEGPGGE(SEQ ID NO.:24)、

[0085] FLIGHTINGIGOGOGGEGPGGE(SEQ ID NO.:25)、

[0086] Ac-IIIIIIGSIIGPGGEGOGGV(SEQ ID NO:26)、

[0087] FLIVIGSIIGPGGEGOGGV(SEQ ID NO.:27)、

[0088] IVIVIGSGIIGPGGEGPGGV(SEQ ID NO.:28)、

[0089] IIIIIGOGIIGOGGEGPGGV(SEQ ID NO.:29)、

[0090] IVIVIGSIIGPGGEGOGGV(SEQ ID NO.:30)、

[0091] LLLLLGSVLGAGEGAGE(SEQ ID NO.:31)、

[0092] LLLLLGPOGGGAGEGAGE(SEQ ID NO.:32)、

[0093] LLLLLGPOGVGPAGEGAGE(SEQ ID NO.:33)、

[0094] LLLLLGPOGIGPAGEGAGE(SEQ ID NO.:34).

[0095] The self-assembly peptides of the present invention having the above-described structure can be initiated by a positively charged source substance to form a liquid three-dimensional network scaffold material under conditions of low concentration and pH > 6. Specifically, the self-assembly peptides are initiated by a positively charged source substance to form a liquid scaffold material under initiation conditions of a concentration of less than 0.1 wt.% and a pH value greater than 6.

[0096] The three-dimensional mesh scaffold material has a nanostructure.

[0097] In some embodiments, the positive charge source material includes a substance with positively charged groups or positively charged ions.

[0098] In some embodiments, the positive charge source substance is a biomolecule, drug, functional molecule, metal ion, amino acid, or a mixture containing one or more of the above components, in which the number of hydrogen bond acceptors is less than the number of hydrogen bond donors.

[0099] In some embodiments, the biomacromolecules include, but are not limited to, organic acids, proteins, polysaccharides, and their derivatives. The organic acids include, but are not limited to, lactic acid, tannic acid, and citric acid. In some embodiments, the proteins include, but are not limited to, fibrinogen, albumin, globulin, hemoglobin, and transferrin. In some embodiments, the polysaccharides and their derivatives include, but are not limited to, chitin and chitosan.

[0100] In some implementations, the drug includes, but is not limited to, antibiotics, dopamine, etc.; preferably, the antibiotic includes, but is not limited to, kanamycin, gentamicin, etc.

[0101] In some implementations, the functional molecules include, but are not limited to, antioxidants, cell proliferation promoting components, etc.

[0102] In some implementations, the antioxidants include, but are not limited to, vitamins such as nicotinamide.

[0103] In some embodiments, the cell proliferation promoting components include, but are not limited to, spermine, spermidine, etc.

[0104] In some embodiments, the metal ions include, but are not limited to, sodium ions, potassium ions, calcium ions, or magnesium ions.

[0105] In some embodiments, the amino acid includes, but is not limited to, amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, etc.

[0106] In some embodiments, the mixture containing the positively charged source substance is serum, plasma, cell culture medium, animal or plant tissue fluid, or a mixture containing the aforementioned positively charged source substance.

[0107] The cell culture media include, but are not limited to: complete cell culture media, cell culture media free of animal source, cell culture media free of animal protein, and cell chemical component-defined culture media.

[0108] In some implementations, the drug is a substance having at least one of the following functions: hemostasis, anti-inflammation, antimicrobial, antifungal, antiviral, antimycoplasma, anticoagulation, analgesia, and promotion of cell, organ, and tissue growth and development.

[0109] In some embodiments, the positive charge source material is a mixture or mixed system comprising one or more of lysine, polylysine, gentamicin, magnesium chloride, fibrinogen, albumin, globulin, hemoglobin, tissue fluid, and serum.

[0110] In some implementations, the positive charge source material is a mixture of animal tissue fluid.

[0111] The self-assembled liquid three-dimensional network fiber structure can bind at least one of the following substances: biochemical molecules with positive electrochemical groups, drugs, functional molecules, animal tissue fluid, and cell culture storage or drug and functional molecule delivery systems containing the above components.

[0112] In some embodiments, the concentration of the self-assembled peptide is from 0.0001 wt.% to 0.1 wt.%, preferably 0.025 wt.% to 0.06 wt.%, and more preferably 0.025 wt.% to 0.05 wt.%.

[0113] In some embodiments, the pH value is 6-10, preferably 6-8.5, and more preferably 6.5-8.

[0114] In some embodiments, the charge concentration ratio of the self-assembled peptide to the positively charged source substance is 1-100:1-100.

[0115] In some embodiments, the positive charge source material can be dissolved in a solvent to obtain a solution, and the self-assembled peptide-positive charge source material solution obtained by mixing the solution with the self-assembled peptide solution has a pH of 6-10, preferably 6.5-8.0, more preferably 6.5-7.5, and even more preferably 7.0-7.5.

[0116] The principle behind the self-assembled peptides described in this invention, which respond to different positively charged source substances to form solution-based scaffold materials, is consistent.

[0117] In a second aspect, the present invention provides a method for forming a scaffold material from a liquid positively charged responsive self-assembled peptide of the first aspect, the method comprising the step of initiating the formation of a liquid scaffold material from the self-assembled peptide using a positively charged source substance under initiation conditions of a concentration of less than 0.1 wt.% of the self-assembled peptide and a pH value greater than 6.

[0118] In some embodiments, the positive charge source material includes a substance with positively charged groups or positively charged ions.

[0119] In some embodiments, the positive charge source substance is a biomolecule, drug, functional molecule, metal ion, amino acid, or a mixture containing one or more of the above components, in which the number of hydrogen bond acceptors is less than the number of hydrogen bond donors.

[0120] In some embodiments, the biomacromolecules include, but are not limited to, organic acids, proteins, polysaccharides, and their derivatives. The organic acids include, but are not limited to, lactic acid, tannic acid, and citric acid. In some embodiments, the proteins include, but are not limited to, fibrinogen, albumin, globulin, hemoglobin, and transferrin. In some embodiments, the polysaccharides and their derivatives include, but are not limited to, chitin and chitosan.

[0121] In some implementations, the drug includes, but is not limited to, antibiotics, dopamine, etc.; preferably, the antibiotic includes, but is not limited to, kanamycin, gentamicin, etc.

[0122] In some implementations, the functional molecules include, but are not limited to, antioxidants, cell proliferation promoting components, etc.

[0123] In some implementations, the antioxidants include, but are not limited to, vitamins such as nicotinamide.

[0124] In some embodiments, the cell proliferation promoting components include, but are not limited to, spermine, spermidine, etc.

[0125] In some embodiments, the metal ions include, but are not limited to, potassium ions, calcium ions, or magnesium ions.

[0126] In some embodiments, the amino acid includes, but is not limited to, amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, etc.

[0127] In some embodiments, the mixture containing the positively charged source substance is serum, plasma, cell culture medium, animal or plant tissue fluid, or a mixture containing the aforementioned positively charged source substance.

[0128] The cell culture media include, but are not limited to: complete cell culture media, cell culture media free of animal source, cell culture media free of animal protein, and cell chemical component-defined culture media.

[0129] In some implementations, the drug is a substance having at least one of the following functions: hemostasis, anti-inflammation, antimicrobial, antifungal, antiviral, antimycoplasma, anticoagulation, analgesia, and promotion of cell, organ, and tissue growth and development.

[0130] In some embodiments, the positive charge source material is a mixture or mixed system comprising one or more of lysine, polylysine, gentamicin, magnesium chloride, fibrinogen, albumin, globulin, hemoglobin, tissue fluid, and serum.

[0131] In some implementations, the positive charge source material is a mixture of animal tissue fluid.

[0132] The self-assembled liquid three-dimensional network fiber structure can bind at least one of the following substances: biochemical molecules with positive electrochemical groups, drugs, functional molecules, animal tissue fluid, and cell culture storage or drug and functional molecule delivery systems containing the above components.

[0133] In some embodiments, the concentration of the self-assembled peptide is 0.0001 wt.% to 0.1 wt.%, preferably 0.025 wt.% to 0.06 wt.%, and more preferably 0.025 wt.% to 0.05 wt.%.

[0134] In some embodiments, the pH value is 6-10, preferably 6-8.5, and more preferably 6.5-8.

[0135] In some embodiments, the charge concentration ratio of the self-assembled peptide to the positively charged source substance is 1-100:1-100.

[0136] In some embodiments, the positive charge source material can be dissolved in a solvent to obtain a solution, and the self-assembled peptide-positive charge source material solution obtained by mixing the solution with the self-assembled peptide solution has a pH of 6-10, preferably 6.5-8.0, more preferably 6.5-7.5, and even more preferably 7.0-7.5.

[0137] The self-assembled peptide solution can be dissolved in neutral or alkaline solvents, and the solution can be adjusted after dissolution. The solvent includes one or more aqueous solutions of sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonia, etc., which can provide an alkaline environment.

[0138] The solvent can be used to dissolve the self-assembled peptides involved in this invention, as long as it can provide a neutral or alkaline environment; a physiologically acceptable solution is preferred.

[0139] The interaction between the self-assembled peptide and the positively charged source substance described in this invention is consistent with the formation principle of the three-dimensional mesh scaffold material involved in this invention.

[0140] Thirdly, the present invention provides a liquid three-dimensional mesh scaffold material, the liquid three-dimensional mesh scaffold material comprising the liquid positive charge responsive self-assembling peptide of the first aspect.

[0141] In some implementations, the liquid three-dimensional mesh scaffold material is obtained using the method of the second aspect.

[0142] In some embodiments, the liquid three-dimensional mesh scaffold material is in solution form.

[0143] In some embodiments, the liquid three-dimensional mesh scaffold material is in the form of an injectable solution.

[0144] In some implementations, the liquid three-dimensional mesh scaffold material is a nanostructure.

[0145] The liquid three-dimensional mesh scaffold material has more β-sheet structures compared to self-assembled peptides.

[0146] In some implementations, the liquid scaffold material solution can effectively disperse cells, microcarriers, positively charged substances, and systems containing one or more of these.

[0147] Fourthly, the present invention provides a composition comprising a liquid positive charge responsive self-assembling peptide of the first aspect and a positive charge source substance of the second aspect.

[0148] The composition is either combined or combined, the latter being that the self-assembled peptide and the positive charge source substance are placed in separate containers.

[0149] In some embodiments, the positive charge source material includes a substance with positively charged groups or positively charged ions.

[0150] In some embodiments, the positive charge source substance is a biomolecule, drug, functional molecule, metal ion, amino acid, or a mixture containing one or more of the above components, in which the number of hydrogen bond acceptors is less than the number of hydrogen bond donors.

[0151] In some embodiments, the biomacromolecules include, but are not limited to, organic acids, proteins, polysaccharides, and their derivatives. The organic acids include, but are not limited to, lactic acid, tannic acid, and citric acid. In some embodiments, the proteins include, but are not limited to, fibrinogen, albumin, globulin, hemoglobin, and transferrin. In some embodiments, the polysaccharides and their derivatives include, but are not limited to, chitin and chitosan.

[0152] In some implementations, the drug includes, but is not limited to, antibiotics, dopamine, etc.; preferably, the antibiotic includes, but is not limited to, kanamycin, gentamicin, etc.

[0153] In some implementations, the functional molecules include, but are not limited to, antioxidants, cell proliferation promoting components, etc.

[0154] In some implementations, the antioxidants include, but are not limited to, vitamins such as nicotinamide.

[0155] In some embodiments, the cell proliferation promoting components include, but are not limited to, spermine, spermidine, etc.

[0156] In some embodiments, the metal ions include, but are not limited to, sodium ions, potassium ions, calcium ions, or magnesium ions.

[0157] In some embodiments, the amino acid includes, but is not limited to, amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, etc.

[0158] In some embodiments, the mixture containing the positively charged source substance is serum, plasma, cell culture medium, animal or plant tissue fluid, or a mixture containing the aforementioned positively charged source substance.

[0159] The cell culture media include, but are not limited to: complete cell culture media, cell culture media free of animal source, cell culture media free of animal protein, and cell chemical component-defined culture media.

[0160] In some implementations, the drug is a substance having at least one of the following functions: hemostasis, anti-inflammation, antimicrobial, antifungal, antiviral, antimycoplasma, anticoagulation, analgesia, and promotion of cell, organ, and tissue growth and development.

[0161] In some embodiments, the positive charge source material is a mixture or mixed system comprising one or more of lysine, polylysine, gentamicin, magnesium chloride, fibrinogen, albumin, globulin, hemoglobin, tissue fluid, and serum.

[0162] In some implementations, the positive charge source material is a mixture of animal tissue fluid.

[0163] The self-assembled liquid three-dimensional network fiber structure can bind at least one of the following substances: biochemical molecules with positive electrochemical groups, drugs, functional molecules, animal tissue fluid, and cell culture storage or drug and functional molecule delivery systems containing the above components.

[0164] In some implementations, the positive charge source material is a mixture of animal tissue fluid.

[0165] This invention unexpectedly reveals that the formed three-dimensional network scaffold structure is more stable under various initiator conditions. Therefore, such self-assembled peptides are particularly suitable for in vivo applications, offering unparalleled advantages over other self-assembled peptides in the prior art. The three-dimensional network scaffold material is a nanostructure.

[0166] Fifthly, this invention provides the application of the liquid positively charged responsive self-assembling peptide of the first aspect, the method of the second aspect, the liquid three-dimensional mesh scaffold material of the third aspect, or the composition of the fourth aspect in one or more of the following: combined application with cell culture microcarriers in cell proliferation; regenerative medicine and tissue regeneration; 2D and 3D cell culture and storage; drug delivery; wound healing; implantable materials; gene therapy; stem cell therapy.

[0167] The liquid scaffold material of the present invention is safe and convenient to prepare, and does not require adjustment of the pH value, temperature, light, salt or ionic composition of the system. It can be self-assembled into a liquid scaffold material by common initiating substances in the biomedical field, which better ensures the biocompatibility of the liquid scaffold material of the present invention.

[0168] The liquid positively charged responsive self-assembling peptide and the liquid three-dimensional mesh scaffold material prepared therefrom provided by this invention can be used for in vitro three-dimensional culture and storage, establishing cell models, loading cells, organs, or organoids, and injecting them into animals or humans for tissue repair; they can also be used as wound dressings, hemostatic materials, etc.; or as carriers for sustained release of drugs or functional factors; or as cell preservation materials in biotherapy, tissue engineering, and regenerative medicine. In summary, the liquid positively charged responsive self-assembling peptide and the liquid three-dimensional mesh scaffold material prepared therefrom of this invention have a wide range of applications and are safe and convenient.

[0169] This invention provides a solution system that, under neutral conditions (i.e., under human physiological conditions), responds to commonly found positively charged ion / group source substances in the body or in cell culture media, forming a three-dimensional nanofiber network structure containing self-assembled peptides. It offers the following advantages: 1. The self-assembled materials involved in this invention are always in a neutral liquid state during use, avoiding the risks associated with adjusting pH or introducing other substances (such as certain metal salt ions, specific proteins, etc.); 2. Because the application state is liquid, it facilitates the dispersion and separation of cells and functional components, the addition of nutrients, the stirring of the culture medium, and injection coating operations; 3. The three-dimensional network scaffold material formed by the self-assembled peptides under positively charged ion / group stimulation has good biocompatibility, a simple preparation method, and broad application prospects and research value in cell culture, tissue engineering, regenerative medicine, and drug delivery systems. Attached Figure Description

[0170] Figure 1 A-1B are images of the self-assembled peptides of the present invention and the three-dimensional mesh scaffold material formed by the self-assembled peptides under the stimulation of a positively charged source substance. (A) 0.1 wt.% self-assembled peptide solution; (B) self-assembled peptide and lysine complex system with a peptide concentration of 0.1 wt.%.

[0171] Figure 2A-2I are FESEM images of the self-assembled peptides and the three-dimensional mesh scaffold material formed by the self-assembled peptides under the stimulation of positively charged source substances of the present invention. (A) is the self-assembled peptide material, (B) is the self-assembled peptide-polylysine complex system, (C) is the self-assembled peptide-fibrinogen complex system, (D) is the self-assembled peptide-gentamicin complex system, (E) is the self-assembled peptide-magnesium ion complex system, (F) is the self-assembled peptide-lysine complex system, (G) is the self-assembled peptide-albumin complex system, (H) is the self-assembled peptide-hemoglobin complex system, and (I) is the self-assembled peptide-globulin complex system.

[0172] Figure 3 A-3I are TEM images of the self-assembled peptides and the three-dimensional mesh scaffold material formed by the self-assembled peptides under the stimulation of positively charged source substances of the present invention. (A) is the self-assembled peptide material, (B) is the self-assembled peptide-polylysine complex system, (C) is the self-assembled peptide-fibrinogen complex system, (D) is the self-assembled peptide-gentamicin complex system, (E) is the self-assembled peptide-magnesium ion complex system, (F) is the self-assembled peptide-lysine complex system, (G) is the self-assembled peptide-albumin complex system, (H) is the self-assembled peptide-hemoglobin complex system, and (I) is the self-assembled peptide-globulin complex system.

[0173] Figure 4 A-4I are circular dichroism spectra of the self-assembled peptides and the three-dimensional mesh scaffold material formed by the self-assembled peptides under the stimulation of positively charged source substances of the present invention. (A) is the self-assembled peptide material, (B) is the self-assembled peptide-polylysine complex system, (C) is the self-assembled peptide-fibrinogen complex system, (D) is the self-assembled peptide-gentamicin complex system, (E) is the self-assembled peptide-magnesium ion complex system, (F) is the self-assembled peptide-lysine complex system, (G) is the self-assembled peptide-albumin complex system, (H) is the self-assembled peptide-hemoglobin complex system, and (I) is the self-assembled peptide-globulin complex system.

[0174] Figure 5 A-5I represent the rheological experimental results of the self-assembled peptides and the three-dimensional mesh scaffold material formed by the self-assembled peptides under the stimulation of positively charged source substances of the present invention. (A) is the self-assembled peptide material, (B) is the self-assembled peptide-polylysine complex system, (C) is the self-assembled peptide-fibrinogen complex system, (D) is the self-assembled peptide-gentamicin complex system, (E) is the self-assembled peptide-magnesium ion complex system, (F) is the self-assembled peptide-lysine complex system, (G) is the self-assembled peptide-albumin complex system, (H) is the self-assembled peptide-hemoglobin complex system, and (I) is the self-assembled peptide-globulin complex system.

[0175] Figure 6This study demonstrates the influence of different sequence structures of self-assembled peptides on the support of erythrocytes in the formation of a three-dimensional mesh scaffold.

[0176] Figure 7 These are comparative images showing how the three-dimensional mesh scaffold material formed by the self-assembled peptides of this invention in response to positively charged source substances provides support for cells. (A) is the control group without self-assembled peptides, (B) is the tube with only self-assembled peptides, (C) is the tube with self-assembled peptides and lysine, (D) is the tube with self-assembled peptides and gentamicin, (E) is the tube with self-assembled peptides and polylysine, and (F) is the tube with self-assembled peptides and fibrinogen.

[0177] Figure 8 A-8C are confocal laser scanning electron microscope images comparing the three-dimensional network scaffold material formed by the self-assembled peptides of this invention in response to positively charged source substances, which provides support for cells. (A) Control group without added self-assembled peptides and positively charged source substances. (B) Three-dimensional network scaffold material formed by the self-assembled peptides in response to tissue fluid supports the three-dimensional distribution of cells. (C) Three-dimensional network scaffold material formed by the self-assembled peptides in response to serum supports the three-dimensional distribution of cells.

[0178] Figure 9 A-9F are confocal laser scanning electron microscope images comparing cells cultured for 4 days after the three-dimensional mesh scaffold material formed by the self-assembled peptides of this invention responding to positively charged source substances. (A) is the control group without the addition of self-assembled peptides, and (BF) are cells cultured for 4 days after the three-dimensional mesh scaffold material formed by SEQ ID NO.:3, SEQ ID NO.:14, SEQ ID NO.:13, SEQ ID NO.:15, and SEQ ID NO.:27 with a peptide concentration of 0.01 wt.% responding to the complete culture medium.

[0179] Figure 10 A-10C are bright-field microscopy comparison images of cells cultured for 5 days in the three-dimensional mesh scaffold material formed by the self-assembled peptide of the present invention in response to a positively charged source substance. (A) is the control group cells cultured for 5 days without the addition of the self-assembled peptide. (B) is the cells cultured for 5 days in the three-dimensional mesh scaffold material formed by the self-assembled peptide SEQ ID NO.:18 of the present invention in response to a positively charged source substance and a complete culture medium. (C) is the cells cultured for 5 days in the three-dimensional mesh scaffold material formed by the self-assembled peptide SEQ ID NO.:19 of the present invention in response to a positively charged source substance and a complete culture medium. The scale bar in the figure is 50 μm.

[0180] Figure 11 A-11B is a schematic diagram of the cell compatibility of the present invention. (A) is a growth curve of cells cultured for 4 days using a three-dimensional mesh scaffold material formed by the response of the self-assembled peptides of the present invention with different peptide concentrations to a positively charged source substance. (B) is a bar chart of the relative viability of cells cultured for 5 days.

[0181] Figure 12 A-12F are confocal laser scanning electron microscope images of three-dimensional cultured cells in a three-dimensional mesh scaffold material formed by the self-assembled peptides of this invention in response to a positively charged source substance. The inset is a magnified view of a representative cell cluster in the corresponding system. The self-assembled peptides used in each experimental group are (A) SEQ ID NO.:3, (B) SEQ ID NO.:21, (C) SEQ ID NO.:23, (D) SEQ ID NO.:25, (E) SEQ ID NO.:2, and (F) SEQ ID NO.:17.

[0182] Figure 13 Cell survival curves for 3D storage of mouse mesenchymal stem cells using the self-assembled peptides of this invention.

[0183] Figure 14 A-14E demonstrates the suspension and support effect of self-assembled peptides on microcarriers, and the proliferation culture of mouse interstitial stem cells using this system. (A) shows polystyrene microcarriers uniformly and stably distributed in the self-assembled peptide solution of SEQ ID NO.:29; (B) shows macroporous gelatin microcarriers uniformly and stably distributed in the self-assembled peptide solution of SEQ ID NO.:30; (C) shows polylactic acid microspheres uniformly and stably distributed in the self-assembled peptide solution of SEQ ID NO.:26; (D) shows a fluorescence microscopy image of the proliferating mouse interstitial stem cells; and (E) shows the cell counting results of the proliferating mouse interstitial stem cells. Detailed Implementation

[0184] This invention achieves self-assembled peptides through ingenious control of the secondary structure of peptides, unique selection of hydrophobic amino acids in the hydrophobic domain, unique selection of hydrophilic amino acids in the hydrophilic domain, and balance and precise coordination of hydrophobic and hydrophilic amino acids.

[0185] This invention provides a liquid positive charge responsive self-assembly peptide, and also provides a liquid scaffold material prepared from the liquid positive charge responsive self-assembly peptide under positive charge source material conditions, which is a solution system with a three-dimensional network scaffold structure.

[0186] The liquid positive charge responsive self-assembling peptide of the present invention remains in a neutral liquid state during use under human physiological conditions, avoiding the risks associated with adjusting pH or introducing other exogenous substances, such as certain metal salt ions or specific proteins.

[0187] The self-assembled peptides of this invention exhibit stronger supporting properties due to the presence of hydroxyproline (O) in the β-turn motif of the hydrophilic domain. Furthermore, this invention has found that the presence of hydroxyproline (O) results in stronger supporting properties in the self-assembled peptides compared to proline (P) present in the β-turn motif of the hydrophilic domain.

[0188] The self-assembling peptides of this invention interact with acidic amino acids and positively charged source substances, triggering the self-assembly of peptide aqueous solutions into a peptide solution system. Under neutral conditions, the addition of a positively charged source substance neutralizes the negatively charged anions on the self-assembly peptide molecules, thereby reducing the repulsive forces between the self-assembly peptide molecules. The self-assembly peptide molecules then achieve self-assembly of polypeptide molecules through hydrophobic interactions and hydrogen bonds, ultimately forming a nanoscale three-dimensional network structure.

[0189] This invention provides a liquid positively charged responsive self-assembling peptide that, at extremely low concentrations (below 0.1 wt.%) and under conditions of pH greater than 6, particularly neutral conditions, forms a solution system containing a 3D scaffold structure in the presence of a positively charged source substance. The positively charged source substance includes substances with positively charged groups, metal cations, or substances containing positively charged groups. In other words, under in vivo conditions, the self-assembling peptide can respond to positively charged ions / groups commonly found in vivo or in cell culture media, forming a liquid solution containing a cross-linked network structure. This facilitates applications requiring physiological conditions, such as cell culture and tissue repair, without the need for additional specific substances or alterations to environmental conditions.

[0190] In the self-assembled peptide network scaffold system of the present invention, the self-assembled peptide used is a single self-assembled peptide from the aforementioned polar peptides, or a mixture of multiple self-assembled peptides; under neutral conditions, the self-assembled peptide can be prepared into a self-assembled peptide solution. The self-assembled peptide of the present invention cannot form a three-dimensional network scaffold material under neutral conditions because it contains at least one acidic amino acid, preventing proper molecular arrangement between the self-assembled peptide molecules. Although the hydrophobic interaction between the hydrophobic ends provides the driving force for molecular aggregation, the electrostatic repulsion between molecules hinders a stable and ordered arrangement. Hydrophilic amino acids tend to be exposed in solution, while acidic amino acids are hydrophilic; the rotational structure reduces interference from other side chains near the acidic amino acid. Therefore, the rotational structure is beneficial for the acidic amino acid to function. It can be considered that the rotational structure makes the side chain groups of the acidic amino acid on the self-assembled peptide more active, intensifying intermolecular repulsion, affecting the molecular arrangement, and further increasing the difficulty of forming a three-dimensional network scaffold structure. When self-assembled peptide molecules approach each other during their movement, they are accelerated by electrostatic interactions, increasing the kinetic energy of the entire system. Therefore, in solutions containing only self-assembled peptides, the distribution of these molecules is relatively chaotic. When positively charged ions / groups are present, the carbonyl groups on the self-assembled peptides form electrostatic interactions. The negative charge on the self-assembled peptide molecules is "shielded," reducing electrostatic repulsion between molecules. Hydrophobic interactions and hydrogen bonds cause the molecules to aggregate in an orderly and regular manner, forming a three-dimensional network scaffold material. Since the interaction between the self-assembled peptides and the positively charged source material described in this invention is consistent with the formation principle of the three-dimensional network scaffold material involved in this invention, several sequences are selected to illustrate the technical effects of the self-assembled peptides of this invention.

[0191] The liquid three-dimensional mesh scaffold material of the present invention is formed by self-assembled peptides under the stimulation of a positively charged source substance. The positively charged source substance is one or more substances with positively charged ions or groups, which generate electrostatic interactions with the negatively charged amino acids on the side chains of the self-assembled peptides of the present invention, thereby neutralizing some or all of the negative charges in the peptide solution system.

[0192] The self-assembled peptides of this invention, when mixed with the aforementioned positively charged source material, stimulate the formation of a three-dimensional network scaffold material without requiring any other modifications to the system. This includes no need to change or adjust the system's temperature or pH, nor to introduce any specific chemical components or add any substance (salt ions, proteins, etc.). The self-assembled peptides of this invention form a three-dimensional network scaffold material in the presence of the positively charged source material, exhibiting broad applicability and requiring no, or preferably no, alteration to the system's chemical composition and environmental conditions.

[0193] The liquid three-dimensional mesh scaffold material of this invention is formed by self-assembled peptides under the stimulation of a positively charged source substance. This positively charged source substance is one or more substances carrying positively charged ions or groups, which generate electrostatic interactions with the negatively charged amino acids on the side chains of the self-assembled peptides, neutralizing some or all of the negative charge in the peptide solution system. The positively charged source substance of this invention includes substances carrying positively charged groups or positively charged ions, including biomolecules, drugs, functional molecules, or small molecules such as metal ions and amino acids, where the number of hydrogen bond acceptors is less than the number of hydrogen bond donors, or mixtures containing one or more of the above substances. Such substances are ubiquitous in cell culture media, serum, plasma, tissue fluid, and the human internal environment. Examples include biomacromolecules containing electron-withdrawing groups such as amino groups (including proteins such as fibrinogen, albumin, globulin, hemoglobin, and transferrin), organic acids such as lactic acid and citric acid, peptides, amino acids such as lysine and polylysine, polysaccharides and their derivatives such as chitin and chitosan), drugs such as antibiotics such as kanamycin and gentamicin, and dopamine, functional molecules such as antioxidants such as vitamins such as nicotinamide, and cell proliferation-promoting components such as spermine and spermidine.

[0194] The self-assembled peptides of this invention, when mixed with the aforementioned substances, stimulate the formation of a three-dimensional network scaffold material without requiring any other alterations to the system. This includes no need to change or adjust the system's temperature or pH, nor to introduce any specific chemical components or add any substance (salt ions, proteins, etc.). The self-assembled peptides of this invention form a three-dimensional network scaffold material in the presence of a positively charged source substance, exhibiting broad applicability and requiring no, or preferably no, alteration to the system's chemical composition and environmental conditions.

[0195] In several embodiments, lysine, gentamicin, magnesium chloride, polylysine, fibrinogen, albumin, globulin, and hemoglobin were used as examples to verify the responsiveness of self-assembled peptides to positively charged ions / groups. The ε-amino group on lysine is highly reactive; the responsiveness of the self-assembled peptides of this invention to lysine means that self-assembled peptides, along with self-assembled peptides containing lysine in their sequence or amino groups on the side chains of their sequence residues, proteins, and mixtures thereof, can also form liquid nanofiber network materials. Gentamicin is a common antibiotic, and its molecule can provide 4-5 amino groups; self-assembled peptides containing gentamicin will provide a liquid nanofiber network material loaded with antibiotics. Magnesium chloride solutions contain free magnesium ions; the responsiveness of the self-assembled peptides of this invention to magnesium ions represents that mixed solutions containing self-assembled peptides and metal ions can form nanofiber network materials. Polylysine has broad-spectrum antibacterial activity, and its molecular surface exposes amino groups attached to the α-carbon (for even stronger activity). The liquid nanofiber network material formed by mixing self-assembled peptides and polylysine is a long-acting broad-spectrum antibacterial material, verifying the feasibility of self-assembled peptides as drug carriers. Fibrinogen is a key factor in blood clotting and tissue repair; albumin is the most abundant protein in human plasma; globulin is a serum protein present in the human body and has immune functions. The response of the self-assembled peptide of this invention to fibrinogen, albumin, globulin, and hemoglobin means that the self-assembled peptide can also form liquid nanofiber network materials with plasma or mixtures containing the above proteins, which is particularly suitable for certain cells with strict culture conditions.

[0196] These are substances commonly found in biological, food, and medical research, and the responsiveness of self-assembled peptides to them strongly demonstrates this. The self-assembled peptides of this invention can directly interact with substances such as drugs, proteins, or cells containing positively charged ions / groups under neutral conditions to form liquid nanofiber network materials. Since the main driving force for this cross-linking is non-covalent bonding, it does not alter the physicochemical properties of the substances providing the positively charged ions / groups.

[0197] In some embodiments, the self-assembled peptides are mixed with lysine, gentamicin, and polylysine to form a three-dimensional mesh scaffold material, meaning that the material can be used for drug delivery, and is particularly suitable for loading biomolecules or synthetic reagents that require physiological conditions.

[0198] This invention provides a three-dimensional mesh scaffold material that enables three-dimensional cell culture. When the self-assembled peptides of this invention are directly mixed with the cell culture medium, the presence of amino-containing biomacromolecules such as albumin, globulin, and hemoglobin in the cell culture medium facilitates cell adhesion and proliferation, forming a three-dimensional mesh scaffold material. The three-dimensional mesh scaffold material mixed with cells remains liquid, allowing for easy stirring or addition of nutrients during culture without the need to change the culture medium. This invention is applicable to various cell types, including hematopoietic cells, stem cells, and cancer cells with stringent growth requirements. The liquid three-dimensional nanofiber network material is also suitable as a hemostatic composition or for promoting wound healing. Because the material is liquid, the peptide solution can be injected or applied to the wound site. The positively charged ions / groups in the patient's blood or tissue fluid, especially various amino-containing proteins such as albumin, globulin, and hemoglobin, enable the self-assembled peptides to respond and form a liquid three-dimensional mesh scaffold material at the wound site, promoting healing. In multiple embodiments, it has been verified that the liquid three-dimensional mesh scaffold material of this invention with a peptide concentration below 0.1 wt.% can be used as a material for three-dimensional cell culture and tissue repair.

[0199] To form a liquid three-dimensional mesh scaffold material, the concentration of the peptide in this invention is preferably below 0.1 wt.%, with a final concentration not less than 0.0001 wt.%, preferably 0.06 wt.% or less, more preferably between 0.025 and 0.06 wt.%, and even more preferably between 0.025 and 0.05 wt.%. The extremely low peptide dosage reduces the cost of using the material. Even at such low peptide concentrations, various applications in the biomedical field can still be achieved, such as cell culture, tissue engineering, regenerative medicine, and drug delivery.

[0200] Unlike other hydrogel materials, the three-dimensional network scaffold material formed by the self-assembled peptides (concentration below 0.1 wt.%) of this invention, under the stimulation of positively charged ions / groups, is in a liquid state. This facilitates the dispersion and separation of cells and functional components, the addition of nutrients, the stirring of culture medium, and injection coating during the operation process. Currently, most peptide or protein materials used and widely studied for cell culture are in the form of hydrogels or gels. When designing these materials, the shear thinning and self-healing properties of hydrogels must be considered, and the gel structure must be disrupted before cell separation. Table 1 lists the main characteristics of the self-assembled peptides and other peptide / protein hydrogel materials of this invention. When using these materials for cell culture, cells should not be added after hydrogelation is complete; it is best to mix them in before self-assembly begins. Therefore, the gel activation conditions should be as compatible as possible with cell growth conditions. (BEAVER NANO) TM 3D cell culture hydrogels involve pH changes during application. The basement membrane matrix requires temperature control to achieve self-assembly. Environmental changes during cell encapsulation can lead to unnecessary apoptosis, placing higher demands on operational conditions. MAX8 hydrogel can achieve gelation initiation under physiological conditions; however, MAX8 hydrogel formation requires a certain concentration of salt ions, which may affect osmotic pressure changes in cells. The initiation factor is no longer limited to a single substance, and the optimal dosage of this material is 0.2-1 wt.%. The self-assembling peptide of this invention responds to positively charged source substances under physiological conditions (pH 7.2-7.4, approximately 37°C) to form a liquid three-dimensional nanofiber network material. Self-assembly initiation does not alter the system's environmental conditions, exhibits broad-spectrum responsiveness to various commonly found substances in vivo, and its optimal concentration is extremely low, at 0.025-0.06 wt.%, several orders of magnitude less than other materials (see Table 1 below).

[0201]

[0202] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0203] Definitions:

[0204] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0205] The alternating hydrophilic and hydrophobic amino acid residues in self-assembling peptides allow them to retain a significant amount of water for self-assembly. The hydrophilic residue side chains interact directly with water, forming inclusion complexes that surround the hydrophobic residue side chains. The number and ratio of hydrophobic and hydrophilic residues in a self-assembling peptide require careful design. Too many hydrophobic residues will render the peptide insoluble in water and cause it to precipitate; conversely, too many hydrophilic residues will result in a highly water-soluble peptide that cannot self-assemble. Furthermore, precise and skillful induction of peptide self-assembly into ordered nanostructures, such as nanofibers, nanotubes, and nanovesicles, is also necessary.

[0206] "Amino acids" include both naturally occurring and non-naturally occurring amino acids, such as D-natural amino acids, β and γ derivatives. According to standard terminology, amino acid residue sequences can be named using three-letter or one-letter codes, for example: alanine (Ala, A); arginine (Arg, R); asparagine (Asp, N); aspartic acid (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 (F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); valine (Val, V); selenocysteine ​​(Sec, U); hydroxyproline (Hyp, O).

[0207] In this invention, "peptide" refers to an amino acid chain. In particular, a peptide is a chain of 2 to 40 amino acids in length.

[0208] In this invention, "self-assembly" refers to the aggregation of peptides into an ordered structure under normal environmental conditions.

[0209] In this invention, "β-sheet" refers to a relatively extended, periodically folded, zigzag-shaped backbone conformation in a self-assembled peptide chain, arranged in parallel or antiparallel configurations to form a β-sheet (sheet). Parallel or antiparallel conformations are determined based on the orientation of the peptide from the N-terminus to the C-terminus. Parallel alignment means that all peptide chains are aligned from the N-terminus to the C-terminus. Antiparallel alignment means that the peptide chains are aligned in opposite directions (i.e., the first peptide chain is aligned from the N-terminus to the C-terminus, while the opposite second peptide chain is aligned from the C-terminus to the N-terminus). Parallel alignment can include peptide translation resulting in the ends of the peptide interleaving. At least half the length of the peptide is involved in interpeptide interactions. In antiparallel alignments, the self-assembled peptides are typically aligned in a line to provide flush ends. This is a typical end-to-end complementary peptide.

[0210] A β-turn is an irregular secondary structure in proteins that causes a change in the orientation of the self-assembled peptide chain. β-turns typically occur at the 180° fold of the peptide chain. A β-turn consists of 3-5 amino acid residues, with the second residue being either proline (P) or hydroxyproline (O). A β-turn motif is generally defined as a turn structure stabilized by a hydrogen bond formed between the carbonyl oxygen atom of the nth amino acid residue and the amide proton of the (n+3)th amino acid residue. β-turns are also called β-bends, reverse bends, or β-loops, and are used to connect β-chains.

[0211] In this invention, "hydrophobicity" refers to a property that tends to repel water or is completely insoluble in water.

[0212] In this invention, "hydrophilicity" refers to the property of easily absorbing water and having a strong polar group that readily interacts with water.

[0213] Hydrophilic amino acids, also known as polar amino acids, have a polar R group that can generally form hydrogen bonds with water molecules, thus exhibiting a certain affinity for water. Hydrophilic amino acids include: S, T, Y, C, U, N, Q, D, E, O, R, K, and H.

[0214] Hydrophobic amino acids, also known as nonpolar amino acids, have a nonpolar R group. They have low or very low affinity for water molecules, but high affinity for lipid-soluble substances. These include amino acids with the following R groups: G, A, V, L, I, P, M, F, and W.

[0215] "Nanostructures" refer to structures with nanoscale dimensions. Nanostructures can be of any shape in one-dimensional, two-dimensional, or three-dimensional space, including nanofilms, nanofibers, nanorods, nanowires, nanofiber networks, nanospheres, nanospirals, and mixtures of several of these. The surface of a nanostructure has a one-dimensional structure at the nanoscale, meaning the surface thickness of the object is between 0.1 nm and 100 nm. Nanotubes have two dimensions at the nanoscale, with diameters ranging from 0.1 to 100 nm and potentially longer lengths. Spherical nanoparticles have three dimensions at the nanoscale, meaning the particle's size in each spatial dimension ranges from 0.1 to 100 nanometers.

[0216] Circular dichroism spectroscopy is the most widely used method for determining the secondary structure of proteins and monitoring conformational changes of protein molecules under external conditions. Because it is performed on a liquid, the results are closer to the secondary structure of proteins under real physiological conditions. It is a rapid, simple and relatively accurate method for studying protein conformation.

[0217] In this invention, both "response" and "initiation" are used to describe the formation of a liquid solution from a self-assembled peptide in response to a positively charged source substance, or the formation of a liquid solution from a self-assembled peptide initiated by a positively charged source substance; the two terms have the same meaning. In this invention, initiator and initiating substance are used interchangeably; they have the same meaning.

[0218] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0219] Example 1: Synthesis of self-assembled peptides

[0220] The self-assembled peptide of the present invention was synthesized by a standard solid-phase self-assembled peptide synthesis method. The amino acid sequence of the self-assembled peptide is shown in the sequence listing SEQ ID NO.:1-34.

[0221] Example 2: Preparation of self-assembled peptide solution

[0222] The self-assembled peptides SEQ ID NO.:1-34 were added to PBS buffer, and alkaline solution was added dropwise until the self-assembled peptides were completely dissolved. The solution was adjusted to neutral 7.2 to obtain a 1 wt.% self-assembled peptide stock solution, which was then autoclaved and stored at 4°C for later use. The peptide concentration was adjusted using this stock solution to obtain self-assembled peptide materials of a predetermined concentration. For example, a certain amount of the self-assembled peptide stock solution was diluted with phosphate buffer to obtain a 0.1 wt.% self-assembled peptide solution.

[0223] Example 3: Preparation of a three-dimensional network scaffold material formed by self-assembled peptides under positive ion / group stimulation

[0224] The self-assembled peptide solution obtained in Example 2 is mixed evenly with the positive charge source substance solution, and the charge concentration ratio of the self-assembled peptide to the positive ion / group source substance in the mixed solution is kept at 1-100:1-100. A three-dimensional network scaffold material formed by the self-assembled peptide under the stimulation of positive ions / groups can be obtained. The pH value of the resulting mixed solution is neutral (about 6 to about 8, preferably about 6.5-7.5, preferably about 7-7.5).

[0225] A mixed solution of self-assembled peptides and positively charged source substances with positive ions / groups was prepared: 0.016 wt.% lysine, 0.016 wt.% gentamicin (calculated based on the presence of 4 amino groups), and 0.012 wt.% magnesium chloride solution (solvent is phosphate buffer, pH is neutral) were mixed with the 0.2 wt.% self-assembled peptide solution obtained in Example 2 (using SEQ ID NO.:3 as an example, the method is the same for other self-assembled peptides) at a volume ratio of 1:1 to obtain a three-dimensional network scaffold material formed by the self-assembled peptides under the stimulation of positive ions / groups. The final concentration of self-assembled peptides in the solution was 0.1 wt.%, and the charge concentration ratio of the self-assembled peptides to the positive ions / groups was approximately 1:1.

[0226] Biological macromolecules such as proteins have relatively large molecular weights and contain a large number of positively charged groups. For ease of calculation, in this embodiment, a mixed solution is prepared with a macromolecule to self-assembled peptide mass ratio of 1:1 (ensuring the charge concentration is 1-100:1-100). Polylysine, fibrinogen, albumin, globulin, and hemoglobin at a concentration of 0.2 wt.% (solvent is phosphate buffer, pH neutral) are mixed with the 0.2 wt.% self-assembled peptide solution obtained in Example 2 (using SEQ ID NO.:3 as an example; the method is the same for other self-assembled peptides) at a volume ratio of 1:1 to obtain a three-dimensional network scaffold material formed by the self-assembled peptides under the stimulation of positively charged ions / groups. The final concentration of the self-assembled peptides in the solution is 0.1 wt.%.

[0227] According to this method, a three-dimensional mesh scaffold material can be configured with an arbitrary predetermined peptide concentration self-assembled peptide and an arbitrary predetermined charge concentration ratio of positive ions / groups as described in this invention.

[0228] Taking lysine as an example, a 0.1 wt.% peptide solution ( Figure 1 A) is a liquid state. When lysine is present, the three-dimensional mesh scaffold material with a peptide concentration of 0.1 wt.% is in a liquid state. Figure 1 B). Other types of positively charged ions / groups stimulate the macroscopic state of the three-dimensional network scaffold material formed by the self-assembled peptides of this invention, which is similar to that of lysine, and is a solution at a peptide concentration of 0.1 wt.%.

[0229] Example 4: Determination of the Material Structure of a Liquid Three-Dimensional Mesh Scaffold

[0230] Experiment 1: Field Emission Scanning Electron Microscopy (FESEM)

[0231] Preparation of self-assembled peptide material samples: The 0.2 wt.% self-assembled peptide material stock solution obtained in Example 2 was diluted with ultrapure water to a final concentration of 0.0001 wt.%, and 10 μL was placed on a superciliary single crystal silicon wafer and allowed to dry at room temperature.

[0232] Preparation of three-dimensional network scaffold material samples formed by self-assembled peptides under positive ion / group stimulation: The self-assembled peptide material stock solution obtained in Example 2 was diluted with deionized water (taking SEQ ID NO.:3 as an example, the method is the same for other self-assembled peptides), positive ion / group solution was added, and mixed evenly to obtain a self-assembled peptide-positive ion / group mixed solution with a final concentration of 0.0001 wt.% of self-assembled peptides at a neutral pH.

[0233] The following describes a method for preparing a self-assembled peptide-positive ion / group mixed solution, using lysine, gentamicin, magnesium chloride, polylysine, fibrinogen, albumin, globulin, and hemoglobin as examples: The 0.2 wt.% self-assembled peptide material stock solution obtained in Example 2 (using SEQ ID NO.:3 as an example; the method is the same for other self-assembled peptides) is diluted with ultrapure water to a self-assembled peptide concentration of 0.0002 wt.%. Lysine, gentamicin, magnesium chloride, polylysine, fibrinogen, albumin, globulin, and hemoglobin are then diluted with ultrapure water to concentrations of 0.000016 wt.%, 0.000016 wt.%, 0.000012 wt.%, 0.0002 wt.%, 0.0002 wt.%, 0.0002 wt.%, 0.0002 wt.%, and 0.0002 wt.%, respectively. The self-assembled peptide solution was mixed with the diluted solution at a volume ratio of 1:1 (the charge concentration ratio of the self-assembled peptide to the positively charged ion / group was approximately 1:1) to obtain a mixed solution of self-assembled peptide-lysine, self-assembled peptide-gentamicin, self-assembled peptide-magnesium ion, self-assembled peptide-polylysine, self-assembled peptide-fibrinogen, self-assembled peptide-albumin, self-assembled peptide-globulin, and self-assembled peptide-hemoglobin. This constitutes a liquid three-dimensional network scaffold material formed by the self-assembled peptides of SEQ ID NO.:3 with a concentration of 0.0001 wt.% under the stimulation of positively charged ions / groups. 10 μL of each solution was placed on a superciliary single-crystal silicon wafer and allowed to dry at room temperature.

[0234] The surface morphology of self-assembled peptide materials and the three-dimensional network scaffold materials formed by self-assembled peptides under positive ion / group stimulation was observed using field emission scanning electron microscopy (FEI-Apreo) at 1 kV. Gold sputtering was performed on the sample surface before observation. The observation results are shown in […]. Figure 2 The self-assembling peptides of this invention can self-assemble into nanofibers under neutral conditions. Figure 2 A), but the fiber distribution is uneven, with obvious differences in fiber length and diameter. A small number of fibers are entangled with another fiber, while most fibers exist as single fibers.

[0235] Different types of positively charged ions / groups all affect the microstructure of self-assembled peptides:

[0236] The presence of polylysine alters the arrangement of the fibers. Figure 2 B) The fibers are arranged in a tight and orderly manner to form fiber bundles, which are intertwined and entangled with each other. This indicates that the self-assembly of the self-assembly peptides in this system is more ordered and stable. The increased length of the fiber bundles makes it difficult to find fine fibers, and the similar diameter of the fiber bundles indicates a more homogeneous system. In systems containing fibrinogen (… Figure 2 C) Gentamicin ( Figure 2 D) Lysine ( Figure 2 F), albumin ( Figure 2 G), hemoglobin ( Figure 2 H), globulins ( Figure 2 In the composite system of I), the presence of fiber bundles and interwoven structures can be clearly observed. The interaction between magnesium ions and self-assembled peptides reduces the differences between fibers. Figure 2 E) The fibers are straight and of similar length, and the fibers interweave to form a network structure.

[0237] Experiment 2: Transmission Electron Microscopy (TEM)

[0238] Preparation of self-assembled peptide material samples: The 0.2 wt.% self-assembled peptide material stock solution (SEQ ID NO.:3) obtained in Example 2 was diluted with ultrapure water to a final concentration of 0.001 wt.%, and 10 μL was placed on a 300-mesh carbon support film copper mesh (Xinxing Bairui) and allowed to dry at room temperature.

[0239] Preparation of three-dimensional network scaffold material samples formed by self-assembled peptides under positive ion / group stimulation: Dilute the 0.2 wt.% self-assembled peptide material stock solution obtained in Example 2 with ultrapure water (taking SEQ ID NO.:3 as an example, the method is the same for other self-assembled peptides), add positive ion / group solution, mix well, and obtain a self-assembled peptide-positive ion / group mixed solution with a final concentration of 0.001 wt.% at pH neutral. Take 10 μL of the mixed solution onto a carbon film copper grid and dry at room temperature.

[0240] The following describes a method for preparing a self-assembled peptide-positive ion / group mixed solution, using lysine, gentamicin, magnesium chloride, polylysine, fibrinogen, albumin, globulin, and hemoglobin as examples: The 0.2 wt.% self-assembled peptide material stock solution obtained in Example 2 is diluted with deionized water to a self-assembled peptide concentration of 0.002 wt.%. Lysine, gentamicin, magnesium chloride, polylysine, fibrinogen, albumin, globulin, and hemoglobin are then diluted with deionized water to concentrations of 0.00016 wt.%, 0.00016 wt.%, 0.00012 wt.%, 0.002 wt.%, 0.002 wt.%, 0.002 wt.%, 0.002 wt.%, and 0.002 wt.%, respectively. The self-assembled peptide solution was mixed with the diluted solution at a volume ratio of 1:1 and incubated at 37°C for one hour to obtain a mixed solution of self-assembled peptides-lysine, self-assembled peptide-gentamicin, self-assembled peptide-magnesium ions, self-assembled peptide-polylysine, self-assembled peptide-fibrinogen, self-assembled peptide-albumin, self-assembled peptide-globulin, and self-assembled peptide-hemoglobin. This represents a three-dimensional mesh scaffold material formed by self-assembled peptides with a concentration of 0.001 wt.% under positive ion / group stimulation. 10 μL of each 0.001 wt.% self-assembled peptide-positive ion / group mixed solution was placed on a 300-mesh carbon support film copper mesh (Xinxing Bairui) and allowed to dry at room temperature.

[0241] Take 10 μL of 2 wt.% phosphotungstic acid negative staining solution and drop it onto the completely dried copper mesh supporting the sample. After 60 seconds, carefully remove the excess staining solution with filter paper. Repeat 3 times. Place the stained copper mesh at room temperature to dry.

[0242] Imaging observation was performed using a Talos G2 200X transmission electron microscope, and the results are as follows: Figure 3 As shown. Similar to the results of experiment (1), different positively charged ions / groups can affect the formation of the three-dimensional network scaffold material of the self-assembled peptides of the present invention.

[0243] Structures formed by self-assembled peptide materials ( Figure 3 A) Composed of interwoven, curved nanofibers with uneven fiber distribution, varying density, and poor orientation. The presence of polylysine reduces the fiber curvature. Figure 3 B), the length is significantly increased, indicating that the self-assembly of the peptides in this system is more ordered and stable. The arrangement of the fibers also changes, with the fibers arranged in a tight and orderly manner to form fiber bundles. These fiber bundles are intertwined and entangled, indicating that the self-assembly peptides of this invention form a three-dimensional network scaffold material under the influence of polylysine. The nano-network structure is beneficial for cell adhesion and three-dimensional culture. Contains hemoglobin (… Figure 3 H), globulins ( Figure 3 The formation of fiber bundles can also be observed in the system of I). When self-assembled peptides encounter gentamicin ( Figure 3 D) and albumin ( Figure 3 G), the increased fiber length and decreased curvature indicate increased structural stability. The interwoven fiber network demonstrates the formation of a three-dimensional mesh scaffold material. Changes in fiber orientation are also observed in the fibrinogen, lysine, and magnesium ion experimental groups ( Figure 3 C, E, F) are no longer Figure 3 The disordered, curved fibers in A indicate that the self-assembling peptides tend to assemble into fibers in a certain direction, which means that the stability of the system increases.

[0244] Combining Experiments 1 and 2, it can be determined that the interaction between the self-assembled peptide and positively charged ions / groups promotes the formation of a three-dimensional network scaffold material. This response of the self-assembled peptide does not change the solution state of the system, but it induces the formation of a three-dimensional network structure. Electron microscopy reveals that the presence of positively charged ions / groups alters the self-assembly path of the self-assembled peptide, resulting in a significantly different microstructure. This not only proves that the self-assembled peptide of this invention forms a three-dimensional nanomatrix in the presence of positively charged ions / groups, but also that this change in fiber morphology and arrangement enhances the structural stability of the liquid nanofiber network material. Experiments 1 and 2 are merely illustrative. When the concentration of the self-assembled peptides of this invention is below 0.1 wt.%, such as 0.06 wt.% or below, between 0.025 and 0.06 wt.%, or below 0.02 wt.%, and when the self-assembled peptides used are SEQ ID NOs:1-7 and SEQ ID NOs:10-34, or conform to the self-assembled peptide structure described in this invention, similar experimental results as in Experiments 1 and 2 can be obtained under the stimulation of positively charged ions / groups. These will not be listed in detail here.

[0245] Experiment 3: Circular Dichroism (CD) Analysis

[0246] Preparation of self-assembled peptide material samples: The 0.2 wt.% stock solution of self-assembled peptide material (SEQ ID NO.:3) obtained in Example 2 was diluted with phosphate buffer solution (0.0067M PO4, pH=7-7.2, Hyclone) to a final concentration of 0.01 wt.% for the self-assembled peptide.

[0247] Preparation of three-dimensional network scaffold material samples formed by self-assembled peptides under positive ion / group stimulation: The 0.2 wt.% self-assembled peptide material stock solution obtained in Example 2 was diluted with phosphate buffer solution, and positive ion / group solution was added at a charge concentration ratio of about 1:1 to obtain a self-assembled peptide-positive ion / group mixed solution with a final concentration of 0.01 wt.% of self-assembled peptides at pH neutral.

[0248] The following describes a method for preparing a three-dimensional network scaffold material formed by self-assembled peptides under positive ion / group stimulation, using lysine, gentamicin, magnesium chloride, polylysine, fibrinogen, albumin, globulin, and hemoglobin as examples: The 0.2 wt.% self-assembled peptide material stock solution obtained in Example 2 is diluted with phosphate buffer solution to a self-assembled peptide concentration of 0.02 wt.%. Lysine, gentamicin, magnesium chloride, polylysine, fibrinogen, albumin, globulin, and hemoglobin are diluted with phosphate buffer solution to concentrations of 0.0016 wt.%, 0.0016 wt.%, 0.0012 wt.%, 0.02 wt.%, 0.02 wt.%, 0.02 wt.%, 0.02 wt.%, and 0.02 wt.%, respectively. The self-assembled peptide solution is mixed with the diluted solution at a volume ratio of 1:1 to obtain a mixed solution of self-assembled peptide-lysine, self-assembled peptide-gentamicin, self-assembled peptide-magnesium ion, self-assembled peptide-polylysine, self-assembled peptide-fibrinogen, self-assembled peptide-albumin, self-assembled peptide-globulin, and self-assembled peptide-hemoglobin with a final concentration of 0.01 wt.%.

[0249] Circular dichroism spectroscopy was performed at room temperature using a MOS-450 / AF-CD spectrometer (Bio-Logic, Claix, France). Samples were placed in a rectangular quartz cell with a 1 mm optical path length. Wavelengths from 190 to 260 nm were measured at a resolution of 0.5 nm and a scan rate of 0.5 nm / s. Solvent background was subtracted, and each sample was scanned three times. The average spectrum was smoothed using a 15-smooth window.

[0250] Circular dichroism spectroscopy results verified the responsiveness of the self-assembled peptides of this invention to positively charged ions / groups, and explained the mechanism of the formation of the three-dimensional network scaffold material from the perspective of molecular structure. Experiments 1 and 2 both indicate that simple self-assembled peptides can also self-assemble into nanofibers in a neutral environment, but the self-assembled peptide molecules within them mainly aggregate in a randomly coiled secondary structure. Figure 4 A) Cannot form a regular, ordered three-dimensional network scaffold material. Polylysine ( Figure 4 B) and albumin ( Figure 4 G) significantly altered the molecular structure of self-assembled peptides; the classic positive and negative peaks of the β-sheet can be observed in the figure. It contains fibroinogen ( Figure 4 C) Gentamicin ( Figure 4 D) Lysine ( Figure 4 F), hemoglobin ( Figure 4 H), globulins ( Figure 4In step I), positive and negative cotton-effects were observed at approximately 195 nm and 218 nm, respectively, indicating an abundance of β-sheet structures. Furthermore, the β-sheet structures formed by self-assembled peptides in this system are more compact and ordered.

[0251] In the presence of magnesium ions, an α-helical CD pattern was observed. Figure 4 E), the pattern contains a maximum positive peak at 192 nm and two minimum negative peaks at 197 and 205 nm, indicating a structural transition from β-sheet to a superhelical-rich conformation. The CD spectra of the self-assembled peptide with polylysine, magnesium ions, and gentamicin show a blue shift compared to the standard α-helical peptide minimums (208 nm, 222 nm). The helical conformation formed by the self-assembled peptide with polylysine, magnesium ions, and gentamicin may be attributed to the stabilization of the helical structure through hydrogen bonds between the carboxyl and amino groups on the self-assembled peptide and intermolecular non-covalent interactions. Circular dichroism spectroscopy analysis indicates that the self-assembled peptide exhibits responsiveness to different types of positively charged ions / groups, and the composition of the secondary structure changes in the presence of positively charged ions / groups. Consistent with the results observed in the microstructures of Experiments 1 and 2, the presence of positively charged ions / groups alters the molecular arrangement of the self-assembled peptide, forming a three-dimensional network scaffold material.

[0252] The same results can be obtained in SEQ ID NOs:1-7 and SEQ ID NOs:10-34.

[0253] Experiment 4, Rheological Experiment

[0254] Preparation of self-assembled peptide material samples: Low-concentration dynamic oscillation scanning was performed using the 0.1 wt.% self-assembled peptide solution (SEQ ID NO.: 7) obtained in Example 2.

[0255] Preparation of three-dimensional network scaffold material samples formed by self-assembled peptides under positive ion / group stimulation: The self-assembled peptide material stock solution (SEQ ID NO:7) obtained in Example 2 was diluted with phosphate buffer solution, and positive ion / group solution was added at a charge ratio of about 1:1 to obtain a self-assembled peptide-positive ion / group mixed solution with a final concentration of 0.1 wt.% of self-assembled peptides at a neutral pH.

[0256] The following describes a method for preparing a three-dimensional network scaffold material formed by self-assembled peptides under positive ion / group stimulation, using lysine, gentamicin, magnesium chloride, polylysine, fibrinogen, albumin, globulin, and hemoglobin as examples: Lysine, gentamicin, magnesium chloride, polylysine, fibrinogen, albumin, and globulin are dissolved and diluted with phosphate buffer solution to concentrations of 0.08 wt.%, 0.08 wt.%, 0.06 wt.%, 1 wt.%, 1 wt.%, 1 wt.%, 1 wt.%, and 1 wt.%, respectively. Mix the self-assembled peptide material stock solution (SEQ ID NO.:7) obtained in Example 2 with the self-assembled peptide material stock solution (0.2 wt.%) at a volume ratio of 1:1 to obtain a mixed solution of self-assembled peptide-lysine, self-assembled peptide-gentamicin, self-assembled peptide-magnesium ion, self-assembled peptide-polylysine, self-assembled peptide-fibrinogen, self-assembled peptide-albumin, self-assembled peptide-globulin, and self-assembled peptide-hemoglobin with a final concentration of 0.1 wt.%. Storage modulus (G') and loss modulus (G”) of self-assembled peptide samples and three-dimensional mesh scaffold material samples formed by self-assembled peptides under positive ion / group stimulation were measured on a 20 mm plate using a MARS 60 rheometer. To determine the formation rate of the three-dimensional nanomatrix, the self-assembled peptide and positive ion / group mixture solution was placed on the plate immediately after preparation for testing. A 500 μm gap was used, and mineral oil was added at the gap to prevent sample dehydration, and data collection began. Dynamic time-scan experiments (DTS) were performed to monitor the changes in storage modulus (G') and loss modulus (G”) over time (1 Hz frequency, 1% strain) for 30 minutes.

[0257] The storage modulus and loss modulus results obtained by oscillation time scanning show that when the concentration of the self-assembled peptide is 0.1 wt.%, all materials are in a liquid state. Figure 5 The storage modulus and loss modulus (A-5I) showed no significant difference and tended towards 0 Pa. When the peptide concentration in the system was below 0.1 wt.%, it should also be in a liquid state. Although macroscopic changes within the material are not readily apparent, the results of Experiments 1, 2, and 3 have already demonstrated the formation of the three-dimensional mesh scaffold material. Notably, the three-dimensional mesh scaffold material with a peptide concentration below 0.1 wt.% is in a liquid state, which facilitates subsequent applications.

[0258] This experiment is only illustrative. When the concentration of the self-assembled peptide used is below 0.1 wt.%, liquid material is formed under the stimulation of the positively charged source material.

[0259] Example 5: The influence of amino acid sequence structure and initiating components on the physical and functional properties of the peptide self-assembly material of the present invention, verification of the self-assembly mechanism, and application of the liquid three-dimensional mesh scaffold material of the present invention in the biomedical field.

[0260] Experiment 1: Verification of the support of different sequences for red blood cells under liquid conditions

[0261] In this experiment, red blood cells were used as an example to verify the supporting effects of hydroxyproline and alanine in the amino acid sequence on cells.

[0262] Experimental methods: Seq 1 (IIIIIGSIIGPGGDGPGGV) and Seq 2 were prepared with PBS buffer at a concentration of 0.1 wt%.

[0263] 2(IIIIIGSIIGPGGEGPGGV), Seq 3(IIIIIGSIIGOGGEGPGGV), and Seq 7(IIIIIGSIIGOGAEGPGGV) self-assembled peptide stock solutions were sterilized at high temperature.

[0264] Using 2ml clear glass bottles, prepare 0.1wt%, 0.05wt%, and 0.01wt% 2mL self-assembled peptide solutions (containing 0.1wt% fibronectin to a final concentration of 0.1wt%) for each of the four materials, as well as a control group (containing 0.1wt% fibronectin but no self-assembled peptide solution). Add 5×10 9 The erythrocyte stock solution was adjusted to a final concentration of 1×10⁻⁶. 8 Blow and mix thoroughly. Take photos every 4 hours to compare the support effect of different materials on red blood cells.

[0265] Experimental conclusion: Among peptides with similar amino acid sequences, hydroxyproline and alanine have a significant impact on the cell support capacity of scaffold solutions under liquid conditions. For example... Figure 6 Under high concentration conditions (0.1 wt%, 0.05 wt%), all four self-assembled peptide solutions could effectively support a concentration of 1 × 10⁻⁶. 8Red blood cells; however, at low concentration conditions (0.01 wt%), hydroxyproline, alanine, and glutamic acid showed significant effects on cell support ability. The four self-assembling peptide solutions showed obvious sedimentation at 8 hours, 24 hours, 32 hours, and 48 hours respectively. The support ability of the four self-assembling peptides from weak to strong is: Seq 1 (IIIIIGSIIGPGGDGPGGV) < Seq 2 (IIIIIGSIIGPGGEGPGGV) < Seq3 (IIIIIGSIIGOGGEGPGGV) < Seq 7 (IIIIIGSIIGOGAEGPGGV). Based on the scaffold network formed in response to fibronectin, compared with Seq 1, glutamic acid in the hydrophilic region of Seq 2, glutamic acid and hydroxyproline in the hydrophilic region of Seq 3, and glutamic acid, hydroxyproline, and alanine in the hydrophilic region of Seq 7 successively showed increasingly stronger enhancement effects on cell support ability. Hydroxyproline is abundant in animal collagen tissues and can enhance the elasticity and support force of the protein matrix through hydrogen bonds formed by its hydroxyl groups. In this invention, by introducing hydroxyproline, the stability of the β-sheet formed by polypeptide self-assembly can be significantly enhanced through the enhanced hydrogen bond network between polypeptides, thereby improving the stability of the self-assembled scaffold and enhancing the cell support function. At the same time, through the substitution of glycine by a single alanine and the substitution of aspartic acid by glutamic acid in the β-sheet structure in the hydrophilic region, without destroying the β-turn structure in the hydrophilic region, the hydrophobicity of the amino acid side chains can be enhanced, and the stability of the self-assembled material can also be increased through hydrophobic interactions, making the scaffold material more supportive to cells.

[0266] In SEQ ID NO.: 8, by adjusting GO at positions 10-11 in SEQ ID NO.: 7 to OG, the formation conditions of the first β-turn in SEQ ID NO.: 7 were completely destroyed, forming the sequence SEQ ID NO.: 8; it was found that SEQ ID NO.: 8 completely lost the self-assembly response to the initiating substance, and even when the initiating substance was added, it could not support red blood cells. This phenomenon further proved that two consecutive β-turns are the most basic conditions for the self-assembly ability of the self-assembling peptides in this invention. By comparing SEQ ID NO.: 9 (IIIIIGSIIGOGGVGPGGV) with SEQ ID NO.: 3 (IIIIIGSIIGOGGEGPGGV), it was found that when the end of the β-turn was no longer connected to an acidic amino acid, the aqueous solution of the self-assembling peptide no longer responded to the initiating substance, and it could effectively support red blood cells in a neutral aqueous solution; this further verified that at least one acidic amino acid connected to the end of the β-turn in the hydrophilic region of the self-assembling peptide is a prerequisite for the self-assembled material of the self-assembling peptide in this invention to respond to the initiating component.

[0267] Experiment 2, Red Blood Cell Support Assay: Effects of Different Initiating Components

[0268] Experimental method: Take 0.16 mL of erythrocyte stock solution (9.17 × 10⁻⁶) 9 Dilute with 10 mM PBS to 10 mL (9.17 MPa). 7 Take 4.5 mL of red blood cell diluent and add it to a 10 mL round-bottom test tube. Add 4.5 mL of 4 mmol / L Calcein-AM solution and incubate at 37 °C for 20 min. Centrifuge at 4 °C and 1500 r / min for 3 min. Discard the supernatant, resuspend in PBS, and repeat 5 times to obtain approximately 1.5 mL of washed red blood cell resuspension (10 mL after washing). 8 ( / mL), ready for use.

[0269] Experimental results: Red blood cells were diluted to 1×10⁻⁶ by adding PBS buffer to the red blood cell resuspended solution. 7 / mL, mix well and let stand for 4 hours, the results are as follows Figure 7 As shown in tube A

[0270] Add 1 wt.% of the self-assembled peptide SEQ ID NO.:3 solution obtained in Example 2 to bring the final peptide concentration in the system to 0.05 wt.% and the final red blood cell concentration to 1 × 10⁻⁶. 7 / mL, mix well and let stand for 4 hours, the results are as follows Figure 7 As shown in tube B.

[0271] A mixture of the self-assembled peptide SEQ ID NO.:3 obtained in Example 2 and lysine was added to the erythrocyte resuspension. This mixture represents the three-dimensional mesh scaffold material formed by the self-assembled peptide in the presence of lysine, bringing the final peptide concentration in the system to 0.05 wt.%. After mixing and standing, the results are as follows: Figure 7 As shown in the middle C tube.

[0272] A mixture of the self-assembled peptide SEQ ID NO.:3 obtained in Example 2 and gentamicin was added to the erythrocyte resuspension. This mixture represents the three-dimensional mesh scaffold material formed by the self-assembled peptide in the presence of gentamicin, bringing the final peptide concentration in the system to 0.05 wt.%. After mixing and standing, the results are as follows: Figure 7 As shown in the middle D tube.

[0273] A mixture of the self-assembled peptide SEQ ID NO.:3 obtained in Example 2 and polylysine was added to the erythrocyte resuspension. This mixture represents the three-dimensional mesh scaffold material formed by the self-assembled peptide in the presence of polylysine, bringing the final peptide concentration in the system to 0.05 wt.%. After mixing and standing, the results are as follows: Figure 7 As shown in the middle E tube.

[0274] A mixture of the self-assembled peptide SEQ ID NO.:3 obtained in Example 2 and fibrinogen was added to the erythrocyte resuspension. This mixture represents the three-dimensional mesh scaffold material formed by the self-assembled peptide in the presence of fibrinogen, bringing the final peptide concentration in the system to 0.05 wt.%. After mixing and standing, the results are as follows: Figure 7 As shown in the middle F tube.

[0275] The control group, which did not contain added peptides and positively charged ions / groups, showed the following results: Figure 7 As shown in tube A, the red blood cells are concentrated at the bottom of the centrifuge tube.

[0276] Based on the results of Experiment 3 in Example 4, it can be seen that the response of the self-assembled peptide to positive ions / groups is reflected in the change of the secondary structure formed by molecular assembly. This change results in the formation of fibers with different morphologies by the self-assembled peptide, and the distribution pattern of the fibers is altered (Experiments 1 and 2 in Example 4). This microscopic change explains the difference between tube CF and tubes A and B in this experiment. Red blood cells are supported by the three-dimensional nanofibers formed by the response of the self-assembled peptide, thus exhibiting a three-dimensional distribution and avoiding sedimentation and adhesion. Without the self-assembled peptide, due to the overall negative charge of the red blood cell membrane, the lack of positive ions / groups prevents the formation of an effective three-dimensional network scaffold material, thus failing to provide the conditions for cell support.

[0277] The results of Experiment 2 show that the three-dimensional mesh scaffold material formed by the self-assembled peptides of this invention in response to different types of positive ions / groups can be obtained in a neutral environment with simple mixing and can provide support for cells, realizing the three-dimensional distribution of cells in the material, which is beneficial for realizing three-dimensional cell culture.

[0278] This experiment further investigated the supporting effect of different self-assembled peptides on erythrocytes under liquid conditions after interaction with different initiating substances. The self-assembled peptide stock solution with a concentration of 0.2 wt% obtained in Example 2 was mixed thoroughly at a volume ratio of 1:1 to obtain the mixed systems of the examples and comparative examples. The supporting phenomena of erythrocytes after the response of each initiating substance to the self-assembled peptides shown in each sequence are shown in Table 2.

[0279]

[0280]

[0281] Experiment 3: Observe the support of cells by the three-dimensional mesh scaffold material formed by the self-assembled peptides of this invention in response to common positive charge source substances in the biomedical field under a confocal laser scanning electron microscope.

[0282] This experiment used common adherent cells, pluripotent stem cells, and umbilical cord-derived mesenchymal stem cells as cell models, and tissue fluid and serum as positive charge source substances to explore the possibility of using the three-dimensional mesh scaffold material formed by the self-assembled peptides of this invention in response to positive charge source substances as a three-dimensional cell culture or tissue repair material.

[0283] Mesenchymal stem cells were cultured in α-MEM (Minimum Essential Medium α) medium (containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% glutamine) at 37°C under humid conditions of 5% CO2 (unless otherwise specified, all cell culture conditions were 37°C, 5% CO2). After digestion, the cells were collected, washed twice with 1×PBS buffer, and resuspended in 1×PBS buffer to obtain 1×10⁻⁶ cells. 6 Cells / mL cell suspension. Mix the cell suspension with half a volume of Calcein-AM staining solution, incubate at 37°C in the dark for 15 min, and then transfer the stained cells to PBS buffer and the self-assembled peptide of this invention, SEQ ID NO.:20 (FLIVIGOGIIGOGGEGPGGV).

[0284] In the three-dimensional mesh scaffold material formed in response to the positively charged source substance and serum, after mixing, the cell distribution was observed using a confocal laser scanning electron microscope in a glass-bottomed culture dish. In the experimental groups containing self-assembled peptides, the final peptide concentration was 0.0025 wt.%, and the charge concentration ratio of the self-assembled peptide to the positively charged source substance was 1-100:1-100. The experimental results are as follows... Figure 8 As shown.

[0285] Without self-assembling peptides and positively charged substances, mesenchymal stem cells naturally settle to the bottom and cannot maintain a three-dimensional distribution. Figure 8 A). Tissue fluid and serum, as common positively charged sources in the biomedical field, contain a large number of positively charged ions / groups. The self-assembled peptides, when mixed with these substances, yield the three-dimensional mesh scaffold material of this invention, providing support for cells and enabling them to be vertically and uniformly distributed in three-dimensional space. Figure 8 B, 8C).

[0286] This experiment demonstrates that the self-assembled peptides of this invention can respond to various positively charged sources to form a three-dimensional mesh scaffold material, and also showcases the excellent cell support provided by this invention. The material of this invention requires only a relatively low dosage to support the three-dimensional distribution of cells in a liquid state, providing a foundation for its application in three-dimensional cell culture.

[0287] Experiments 1-3 demonstrated the supporting effect of the material of the present invention on cells using different cell models and different observation methods. The material can effectively support the three-dimensional distribution of cells and prevent cells from settling and adhering to the cell wall.

[0288] Experiment 4: HepG2 liver cancer cells were cultured using a three-dimensional mesh scaffold material formed by the response of the self-assembled peptides of this invention to positively charged ions / groups.

[0289] Complete culture media commonly used in in vitro cell culture contain a large number of positively charged ions / groups. As a mixture of various positively charged ions / groups, the three-dimensional mesh scaffold material formed by self-assembled peptides in response to complete culture media is also a type of material of the present invention.

[0290] Using HepG2 hepatocellular carcinoma cells, a common adherent cell line, as a cell model, cells were cultured for 4 days using the self-assembled peptides obtained in Example 2: SEQ ID NO.:3 (IIIIIGSIIGOGGEGPGGV), SEQ ID NO.:14 (IIIIIGTVIGPGGEGOGGE), SEQ ID NO.:13 (IIIIIGOGIIGPGGEGPGGE), SEQ ID NO.:15 (IIIIIGTVIGPGGEGOGGK), and SEQ ID NO.:27 (FLIVIGSIIGPGGEGPGGV). The cell status in the material of this invention was observed. The specific experimental setup was as follows: HepG2 hepatocellular carcinoma cells were cultured in high-glucose DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% fetal bovine serum at 37°C under a humid environment of 5% CO2. The cell seeding density in all groups was 6 × 10⁶ cells / year. 5 The control group underwent two-dimensional culture without peptide buffer, while the experimental groups were cultured with the self-assembled peptides obtained in Example 2 (SEQ ID NO.:3, SEQ ID NO.:14, SEQ ID NO.:13, SEQ ID NO.:15, and SEQ ID NO.:27), respectively, to achieve a final peptide concentration of 0.05 wt.%. Cells from each group were cultured in 6-well plates for four days, after which they were isolated and collected. After washing twice with 1×PBS buffer, the cells were resuspended in PBS to achieve a cell suspension concentration of 1×10⁻⁶. 5 -1×10 6 Cells / mL were used to fluorescently label live and dead cells using the Calcein-AM / PI live / dead cell double staining kit, and then re-inoculated into the corresponding original system. The cells were incubated for 15 min (37°C, humid environment with 5% CO2) and observed under a confocal laser scanning electron microscope.

[0291] Figure 9This shows the state of the cells after four days of culture. Compared to the control group without the addition of self-assembling peptides (… Figure 9 Compared to group A), the experimental group ( Figure 9 In BF culture, all liver cancer cells were distributed three-dimensionally and did not adhere to the culture medium. This indicates that the self-assembled peptides responded to the positively charged ions / groups in the high-glucose DMEM medium used for cell culture, forming a three-dimensional mesh scaffold material to support the three-dimensional culture of liver cancer cells.

[0292] In addition, in the experimental group, the materials were mixed with a complete culture medium containing multiple stimuli such as albumin, globulin, fibrinogen, and lysine under neutral pH conditions to complete the liquid three-dimensional culture of cells. The operation was simple and convenient, requiring no gelation or stirring, and would not cause damage to the cells.

[0293] Experiment 5: Using the three-dimensional mesh scaffold material formed by the response of the self-assembled peptides of this invention to positive ions / groups to culture mesenchymal stem cells.

[0294] Mesenchymal stem cells were cultured in α-MEM medium (containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% glutamine) containing the self-assembling peptide compounds of the present invention, SEQ ID NO.:18 (IIIIIGOGIIGPGGEGPGGV) and SEQ ID NO.:19 (IIIIIGOGIIGPGGDGPGGD), respectively. The above-mentioned medium without the self-assembling peptides was used for cell culture as a control group. The cell seeding density for both was 6 × 10⁶ cells / year. 5 In the groups containing self-assembled peptides, the final peptide concentration was 0.025 wt.% (cells / mL). After 5 days of culture, the cell status was observed using an inverted microscope, and the results are as follows: Figure 10 As shown.

[0295] Mesenchymal stem cells cultured in a complete culture medium without self-assembling peptides adhered to the culture dish and grew. After 5 days, the cell confluence was observed to reach over 80%, and the cells were about to cover the bottom of the culture dish. Figure 10 A); After adding the self-assembled peptide of this invention to the culture medium, the self-assembled peptide forms a three-dimensional mesh scaffold material in response to the α-MEM culture medium. Mesenchymal stem cells no longer grow attached to the surface of the culture dish, but instead grow in a spherical shape, maintaining a three-dimensional distribution and achieving three-dimensional culture. Figure 10(B, 10C). In the 3D experimental group, the three-dimensional nanonetwork formed by the self-assembled peptides of this invention under the stimulation of a positively charged source material—serum-containing α-MEM medium—can simulate the microenvironment of natural cell growth, allowing cells to grow close to their natural state. This provides a foundation for applications such as studying cell-cell interactions, cell migration, and cell-based drug detection. Furthermore, three-dimensional culture significantly expands the cell proliferation space; within the same time frame, cell confluence is lower, and cells can still continuously proliferate within the material, which is beneficial for large-scale cell culture.

[0296] Experiment 6. Cell compatibility of the three-dimensional mesh scaffold material formed by the self-assembled peptides of this invention in response to positive ions / groups.

[0297] To demonstrate the good biocompatibility of the material of this invention, HepG2 liver cancer cells were used as a cell model. HepG2 liver cancer cells were seeded into 6-well plates, with each well containing approximately 50,000 cells. Cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and different concentrations of SEQ ID NO.:3 (IIIIIGSIIGOGGEGPGGV) peptide (0.005 wt.%, 0.01 wt.%, 0.03 wt.%, and 0.05 wt.%), as well as in the same medium without peptide. After uniform dispersion, cells were cultured for 4 days. Cells were collected every 24 hours, counted, and cell growth curves were recorded. All experiments were performed in 5 replicates. Results are shown below. Figure 11 A.

[0298] The changes in cell viability in the material of this invention were tested using the CCK-8 (Cell Counting Kit-8) assay. Cell density was set at 5 × 10⁶ cells / year. 4 HepG2 liver cancer cells were seeded at a concentration of 100 μL / mL into 96-well plates using complete culture medium containing different concentrations of SEQ ID NO.:3 (IIIIIGSIIGOGGEGPGGV) (control group without SEQ ID NO.:3 (IIIIIGSIIGOGGEGPGGV)). After 5 days of incubation, the medium was replaced with fresh complete culture medium containing 10% CCK-8 solution at 24, 48, 72, 96, and 120 hours, and incubated for 30 minutes. The absorbance (OD) at 450 nm was measured using a multi-mode microplate reader. All experiments were performed in 2-5 replicates. The daily relative cell viability was calculated using the following formula:

[0299] V t =(S t -S b ) / (S1-S b )×100%;

[0300] Where V t S represents the relative cell viability over t days. t S is the average OD value measured over t days. b S1 represents the average OD value of the blank background measured over t days, and S2 represents the average OD value measured on the first day.

[0301] The results are as follows Figure 11 As shown in B.

[0302] pass Figure 11 The cell growth curves for group A show that, in the first three days, the number of cells in the control group (without SEQ ID NO.:21) was higher than that in other groups. Combined with Experiment 6, this indicates that cells adhered to the plate without the self-assembling peptide. During this stage, the cells in the control group were in the logarithmic growth phase and grew rapidly. However, the bottom area of ​​the well plate was limited, and adherent cells often exhibited contact inhibition; that is, when the cell confluence reached 100%, cell proliferation ceased and apoptosis began. Therefore, a rapid decrease in cell number was observed on the fourth day. When the system contained SEQ ID NO.:3 (IIIIIGSIIGOGGEGPGGV), peptide concentrations of 0.005-0.05 wt.% allowed cells to proliferate continuously for four days, and the total number of cells exceeded that of the control group on the fourth day. Combined with the results of Experiment 6, it can be seen that in the experimental group system, cells were able to grow in three-dimensional space, and the growth space was greatly expanded, thus ensuring larger-scale cell proliferation over a longer period.

[0303] In the CCK-8 experiment, through Figure 11 As can be seen from B, in the cell culture system containing the self-assembled peptide of the present invention, the cell viability was consistently higher than on the first day (100% on the first day), and the cell viability of all experimental groups on the fifth day was higher than that of the control group. In most experimental groups, the cell viability was observed to increase during the five-day culture process. In summary, the cells maintained good activity in all experimental groups, indicating that the three-dimensional mesh scaffold material formed by the self-assembled peptide of the present invention in response to positive ions / groups can promote cell proliferation and maintain a higher level of cell viability for a longer period of time compared with traditional 2D culture.

[0304] This experiment verified, through changes in cell growth curves and relative viability, that the self-assembled peptides of this invention respond to the complete culture medium commonly used in cell culture to form a three-dimensional mesh scaffold material. This material has good cell compatibility and maintains the continuous proliferation and viability of cells over a longer culture period while achieving three-dimensional cell culture.

[0305] Experiments 4, 5, and 6 used mesenchymal stem cells and liver cancer cells as cell models, demonstrating that the material of this invention has good biocompatibility, supports three-dimensional cell culture, and that the cells maintain high activity and continue to proliferate within the material during culture.

[0306] Experiment 7. Stem cell proliferation within a three-dimensional mesh scaffold material formed by the self-assembled peptides' response to positive ions / groups in this invention.

[0307] Using adherent mesenchymal stem cells (MSCs) as a cell model, the cell proliferation-promoting effect of the material of this invention was verified. MSCs were added to α-MEM medium (containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% glutamine) containing the self-assembled peptide compounds of this invention, SEQ ID NO.:3 (IIIIIGSIIGOGGEGPGGV), SEQ ID NO.:21 (IVIVIGSGIIGPGGDGPGGV), SEQ ID NO.:23 (IIIIIGSIIGPGGEGOGGV), SEQ ID NO.:25 (FLIVI GOGIIGOGGEGPGGE), SEQ ID NO.:2 (IIIIIGSIIGPGGEGPGGV), and SEQ ID NO.:17 (IIIIIGTVIGPGGEGOGGV), respectively. The cell seeding density was 1 × 10⁻⁶ cells / year. 6 Cells / mL, with a final concentration of 0.05 wt.% for all self-assembled peptides. To more clearly observe cell distribution and morphology, cells cultured for 4 days were stained with AM / PI live / dead cells, and cell growth was observed using a laser confocal microscope.

[0308] The results are as follows Figure 12 As shown in A-12F, after adding a complete culture medium containing initiators such as albumin, globulin, fibrinogen, and lysine, the self-assembled peptides of this invention at a concentration of 0.05 wt.% formed a three-dimensional network material. MSCs cultured in this material formed distinct cell clusters after 4 days, achieving liquid 3D cell culture. This indicates that cells cultured within the self-assembled peptide network material of this invention have excellent expansion capabilities. Experiments verified that the three-dimensional network structure formed by the self-assembled peptides of this invention under the stimulation of positively charged ions / groups can serve as a good cell culture material even at extremely low concentrations. The liquid three-dimensional network scaffold material of this invention can simulate the microenvironment of natural cell growth, allowing cells to grow close to their natural state, providing a foundation for applications such as cell-cell interactions, cell migration, and cell-based drug detection.

[0309] Other numbered self-assembling peptides showed similar experimental results, which will not be elaborated here.

[0310] Experiment 8: Application of 3D Cell Storage

[0311] Experimental Methods: This experiment uses the storage of mouse mesenchymal stem cells at 4℃ as an example. After isolation, the mouse mesenchymal stem cells were thoroughly mixed by pipetting, and 10 ml aliquots were transferred into cryovials. The cells were then thoroughly mixed to achieve a cell concentration of 1×10⁻⁶. 6 Approximately 100 cells / mL. In group 3D, mouse serum was added to 1% penicillin antibody in 50% MAP + 50% SFEM stock solution, followed by the addition of peptide SEQ ID NO.:25 (FLIVIGOGIIGOGGEGPGGE) to a final concentration of 0.05%. In group 2D, mouse serum was added to 1% penicillin antibody in 50% MAP + 50% SFEM stock solution, and then the mixture was stored at 4°C.

[0312] Cell viability test:

[0313] (1) Mix the preserved cells thoroughly by pipetting, take 500 μL of cell suspension into 1.5 mL EP tubes, add PBS to wash, and centrifuge at 1500 rpm for 10 min.

[0314] (2) Discard the supernatant, resuspend the cells in 150 μL of 1% PBA solution, then add 150 μL of live and dead cell staining working solution, mix thoroughly, incubate at 37°C for 15 min in the dark, and then detect the cells using flow cytometry.

[0315] Experimental Results: To understand the changes in mesenchymal cell viability during the storage period, we used a Calcein-AM / PI live / dead cell double staining kit for double staining, and then analyzed the results using flow cytometry. Figure 13 As shown, starting from day 3 of storage, the survival rate of mouse mesenchymal stem cells in the 3D storage group was significantly higher than that in the 2D storage group, with a statistically significant difference (79% vs. 67%). Furthermore, a high cell survival rate was still observed on day 5 of storage (71% vs. 43%), an increase of nearly 30%. This indicates that the mesenchymal stem cell storage system of this invention has a better storage effect than traditional 2D storage, greatly improving cell survival rate.

[0316] The experimental results for other self-assembled peptides with different sequence numbers are similar to those in this example, and will not be described in detail here.

[0317] Experiment 9: Application of the scaffold and 3D cell culture microcarrier of the present invention in cell proliferation.

[0318] Experimental Method: The synthesized self-assembled peptide shown in SEQ ID NO.:29 (IIIIIGOGIIGOGGEGPGGV) was dissolved in cell culture medium and vortexed until fully dissolved to obtain a 0.1% self-assembled peptide solution. The pH of the solution was adjusted to 7.4 with 0.1M NaOH solution. Then, it was mixed with polystyrene microcarriers (particle size between 100-500 μm), and 10% fetal bovine serum was added. Finally, it was dispensed to obtain an injectable solution containing 1% microcarriers of the self-assembled peptide mixture. The solution became opaque, and the microspheres were uniformly and stably distributed in the self-assembled peptide solution. Figure 14 A). The resulting self-assembled peptide mixture injection containing microspheres, after being left at room temperature for one month, showed that all the injections were in a uniform suspension state with no obvious solid-liquid separation. The same experimental method was used to support macroporous gelatin microcarriers (particle size between 100-500 μm) using SEQ ID NO.:30 (IVIVIGSIIGPGGEGOGGV). Figure 14 B) Polylactic acid microspheres (particle size between 100-500 μm) supported by SEQ ID NO.:26 (Ac-IIIIIGSIIGPGGEGOGGV) Figure 14 C)

[0319] This experiment further validated the combined use of SEQ ID NO.:29 (IIIIIGOGIIGOGGEGPGGV) and polystyrene microspheres to promote the proliferation of mouse mesenchymal stem cells. Ninth-generation rat bone marrow mesenchymal stem cells (BMSCs) were thawed and cultured in Amem medium supplemented with 10% FBS and 1% penicillin / streptomycin (Gibco). All cells used in this study were at passages 8-15. Before cell seeding, the polystyrene microcarriers were immersed in 70% (v / v) ethanol for 1 hour, followed by exposure to UV light for 30 minutes. The polystyrene microcarriers were incubated in medium for 12 hours before cell seeding. BMSCs were seeded and incubated at 37°C in a humid environment containing 5% CO2. Cells were harvested with trypsin containing EDTA after reaching 80% confluence. Cells were seeded in TC-free 48-well plates at 1-2 × 10⁶ mg / well. 4 Cells were seeded at a density of 1 wt% with microcarriers, and a self-assembled peptide scaffold was added on day 2 of culture to achieve 3D culture. Every two days, 80% of the culture medium was withdrawn and replaced with an equal volume of fresh culture medium and the self-assembled peptide scaffold. To study cell attachment and growth, samples were collected and cell counts were performed on days 1, 3, 5, 7, and 9 of cell culture. Cells were stained with an AM / PI cell viability staining kit on days 1, 4, and 7 and analyzed using a laser confocal microscope.

[0320] Experimental Results: First, the support of the self-assembled peptides of this invention for polystyrene microcarriers was evaluated under static conditions, thereby verifying the proliferation of mouse mesenchymal stem cells in this composite system. Fluorescence microscopy images under static culture conditions (…) Figure 14 D) indicates that a small number of cells adhered to the polystyrene microcarriers on day 1, demonstrating the strong cell adsorption capacity of the microcarriers. Subsequently, on days 4 and 7 of cell culture, visible cell proliferation was observed, with a significant increase in the number of cells on the microspheres, indicating that the self-assembled peptide 3D scaffold combined with the microcarrier composite system can maintain cell proliferation without cytotoxicity. Cell counting results ( Figure 14 E) also supports this view. After 3 days, the cells in the 2D group showed contact inhibition and stopped proliferating, while the cells in the 3D group remained in a proliferating state, indicating that the 3D culture system is suitable for large-scale stem cell culture.

[0321] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A liquid positive charge responsive self-assembly peptide, wherein the liquid positive charge responsive self-assembly peptide is composed of a hydrophobic domain, a hydrophilic domain, and a linker domain, wherein the linker domain provides a spacer region between the hydrophobic domain and the hydrophilic domain; or the liquid positive charge responsive self-assembly peptide is composed of a hydrophobic domain and a hydrophilic domain; The hydrophilic domain consists of at least two consecutive β-turn regions capable of forming β-turns, and at least one of the β-turn regions contains one or more acidic amino acids at its end; The liquid positive charge responsive self-assembling peptide contains at least one hydroxyproline (O) located in a hydrophilic domain and / or a linker domain; The β-turn region is composed of a β-turn motif formed by 4-6 amino acids, and the β-turn motif has the following structure: X1X2X3X4, X1X2X3X4X5, or X1X2X3X4X5X6, where... X1, X2, X3, X4, X5, and X6 are amino acid residues. In each β-turn motif, X1, X2, X3, X4, X5, and X6 are either the same as or different from each other. The amino acid sequences of the liquid positive charge responsive self-assembled peptide are selected from SEQ ID NOs:3-7, 10~20, 22, 23, 25~27, 29, 30 and SEQ ID NOs:32-34.

2. A method for forming a liquid scaffold material from the liquid positively charged responsive self-assembly peptide of claim 1, the method comprising the step of initiating the formation of a liquid scaffold material solution from the self-assembly peptide using a positively charged source substance under initiation conditions where the pH of the self-assembly peptide solution is greater than 6; wherein the positively charged source substance comprises a substance having a positively charged group or a positively charged ion.

3. The method according to claim 2, wherein the concentration of the self-assembled peptide is from 0.0001 wt.% to 0.1 wt.%. The pH value is 6-10; The charge concentration ratio of the self-assembled peptide to the positively charged source substance is 1-100:1-100.

4. The method according to claim 3, wherein the concentration of the self-assembled peptide is 0.025 wt.% to 0.06 wt.%.

5. The method according to claim 3, wherein the concentration of the self-assembled peptide is 0.025 wt.% to 0.05 wt.%.

6. The method according to claim 3, wherein the pH value is 6-8.

5.

7. The method according to claim 3, wherein the pH value is 6.5-8.

8. The method according to claim 2, wherein the positive charge source substance is a biological macromolecule, drug, functional molecule, metal ion, amino acid, or a mixture containing one or more of the above components, in which the number of hydrogen bond acceptors is less than the number of hydrogen bond donors.

9. The method according to claim 8, wherein the biomacromolecule is selected from organic acids, proteins, polysaccharides and their derivatives.

10. The method according to claim 9, wherein the protein is selected from fibrinogen, albumin, globulin, hemoglobin or transferrin.

11. The method according to claim 9, wherein the polysaccharide and its derivatives are selected from chitin or chitosan.

12. The method according to claim 8, wherein the drug is selected from antibiotics and dopamine.

13. The method according to claim 12, wherein the antibiotic is selected from kanamycin and gentamicin.

14. The method according to claim 8, wherein the functional molecule is selected from antioxidants and cell proliferation promoting components.

15. The method according to claim 14, wherein the antioxidant is selected from vitamin antioxidants.

16. The method according to claim 14, wherein the cell proliferation promoting component is selected from spermine or spermidine.

17. The method according to claim 8, wherein the metal ion is selected from potassium ions, calcium ions, or magnesium ions.

18. The method according to claim 8, wherein the amino acid is selected from lysine, arginine, polylysine, and polyarginine.

19. The method according to claim 8, wherein the positive charge source substance is serum, plasma, cell culture medium, animal or plant tissue fluid, or a mixture of one or more of these.

20. The method according to claim 9, wherein the organic acid is selected from lactic acid, tannic acid or citric acid.

21. A liquid scaffold material comprising the liquid positive charge responsive self-assembling peptide of claim 1, or prepared by the method of any one of claims 2-20.

22. The liquid scaffold material according to claim 21, wherein the liquid scaffold material is a three-dimensional mesh scaffold material in solution form.

23. A composition comprising the liquid positive charge responsive self-assembling peptide of claim 1 and the positive charge source substance of any one of claims 2-20.

24. The use of the liquid positive charge responsive self-assembling peptide of claim 1, the liquid scaffold material prepared by the method of any one of claims 2-20, the liquid scaffold material of claim 21 or 22, or the composition of claim 23, selected from one or more of the following: combined use with cell culture microcarriers in cell proliferation; 3D cell culture and storage.

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