Self-assembling peptides

Abstract

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

Description

Self-assembling peptides Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to peptide hydrogel biomaterials. Background Art

[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 a certain structure under specific responses. For example, these self-assembling peptides can respond to different factors such as temperature, pH, light, metal ions, and certain specific proteins, and then self-assemble to form a network scaffold structure, thereby exerting functions such as cell support, antibacterial, and tissue repair. They can be used in applications such as drug delivery, drug screening and evaluation, antibacterial, tissue engineering, and 3D cell culture. However, these response factors may also impose certain limitations on practical applications and increase the risk of adverse applications. For example, higher or lower pH values ​​(the peptide hydrogel described in CN201680012178.5 needs to be used when pH is less than 3.5) and higher salt ion concentrations can cause damage to cells (the peptide hydrogel used for skin repair in CN202110863352.X requires a specific sodium ion concentration); the introduction of some exogenous initiating substances (CN201810783581.9 involves a peptide composite hydrogel, and the gelation process requires the introduction of calcium phosphate) may cause certain human safety risks in clinical applications; and response factors such as temperature and light (the gelation of the peptide in CN201680061179.9 depends on photoactivation) make the setting of self-assembly initiation conditions a factor that must be considered in practical applications, thereby placing certain restrictions on the application conditions of self-assembling polypeptides. When using these hydrogels in biological experiments, non-physiological temperatures and pH levels pose a significant threat to cell activity; photoresponsive gelation relies on complex and expensive peripheral devices; high metal salt ion concentrations affect the osmotic pressure of the cellular microenvironment; and exogenous substances may induce immune responses, posing certain safety risks. Existing peptides capable of gelation in response to endogenous substances are relatively few in number, and most are limited to a single endogenous substance (such as CN201380063880.0, which involves a peptide-albumin hydrogel), limiting their scope of application.

[0003] Therefore, the development of self-assembling peptides that can respond and self-assemble to form nano-network structures 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 will have very important application prospects.

[0004] Summary of the Invention

[0005] The present invention relates to a self-assembling peptide solution system which can respond to and self-assemble into a nano-network structure under a wide range of endogenous material conditions and can exert supporting and repairing functions in a solution state.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0045] In some embodiments, the hydrophobic domain comprises:

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

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

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

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

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

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

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

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

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

[0055] GPOGI (SEQ ID NO.: 88), GPOGV (SEQ ID NO.: 89), GPOGL (SEQ ID NO.: 90), GSII (SEQ ID NO.: 91), GSGII (SEQ ID NO.: 92), GSVI (SEQ ID NO.: 93), GOII (SEQ ID NO.: 94), OGII (SEQ ID NO.: 95) or GTVI (SEQ ID NO.: 96), wherein S, T, and O are interchangeable with each other.

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

[0057] GSVL (SEQ ID NO.:97), GSII (SEQ ID NO.:91), GTII (SEQ ID NO.:98), GTVI (SEQ ID NO.:96), GOVI (SEQ ID NO.:99), GSVI (SEQ ID NO.:93), GSGII (SEQ ID NO.:92), GSGVI (SEQ ID NO.:100), GOII (SEQ ID NO.:94), OGII (SEQ ID NO.:95), GOGVI (SEQ ID NO.:101) or GOGII (SEQ ID NO.:102).

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

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

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

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

[0062] IIIIIIGSIIGPGGDGPGGV (SEQ ID NO.: 1);

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

[0064] IIIIIGSIIGOGGEGPGGV(SEQ ID NO.:3)

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

[0066] IIIIIGSIIGOGGPGGGGV(SEQ ID NO.:5)

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

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

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

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

[0071] FLIVIGSIIGOGGEGPGGV(SEQ ID NO.:11)

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

[0073] IIIIIGOGIIGPGGEGPGGE(SEQ ID NO.:13)

[0074] FIFIFGTVIGPGGEGOGGV(SEQ ID NO.:14);

[0075] IFIFIGTVIGPGGEGOGGK(SEQ ID NO.:15);

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

[0077] IVIVIGSIIGPGGDGPGGV(SEQ ID NO.:17)

[0078] IVIVIGSIIGOGGDGPGGV(SEQ ID NO.:18)

[0079] IVIVIGSIIGPGGEGOGGV (SEQ ID NO.:19);

[0080] FLIVIGOGIIGOGGEGPGGE (SEQ ID NO.:20);

[0081] IVIVIGIGIIGOGGDGOGGV (SEQ ID NO.:21);

[0082] IVIVIGSGIIGPGGEGPGGV (SEQ ID NO.:22);

[0083] FIIIVGSIIGPGGEGPGGV (SEQ ID NO.:23);

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

[0085] IIIIIIGOGIIGOGGEGPGGV (SEQ ID NO.:25);

[0086] Ac-IIIIGSIIGPGGEGOGGV (SEQ ID NO.:26);

[0087] FLIVIGSIIGOGAEGPGGV (SEQ ID NO.:27);

[0088] FLIVIGSIIGOGAEGOGGV (SEQ ID NO.:28);

[0089] LLLLLSVLGPAGEGPAGE(SEQ ID NO.:29);

[0090] LLLLLGPOGLGPAGEGPAGE(SEQ ID NO.:30);

[0091] LLLLLGPOGVGPAGEGPAGE (SEQ ID NO.: 31); or

[0092] LLLLLGPOGIGPAGEGPAGE (SEQ ID NO.:32).

[0093] The self-assembling peptide of the present invention having the above structure can be triggered by an initiator to form a three-dimensional network scaffold material.

[0094] The three-dimensional mesh scaffold material has a nanostructure.

[0095] The initiator is a positive charge source substance, and the positive charge source substance includes a substance with a positively charged group or a positively charged ion.

[0096] In some embodiments, the positive charge source substance is a biomacromolecule, drug, functional molecule, or small molecule such as metal ions, amino acids, etc., whose number of hydrogen bond acceptors is less than that of hydrogen bond donors under neutral conditions, or a mixture comprising one or more of the above substances.

[0097] In some embodiments, the biomacromolecules include but are not limited to organic acids, proteins, polysaccharides and derivatives thereof, and the organic acids include but are not limited to lactic acid, tannic acid, citric acid and the like.

[0098] In some embodiments, the protein is selected from proteins that are capable of donating hydrogen ions under neutral physiological conditions.

[0099] Preferably, the proteins are independently selected from proteins having an isoelectric point (PI) value lower than 7.0 (preferably 3.4-6.05).

[0100] In some embodiments, the protein includes, but is not limited to, fibrinogen, globulin, hemoglobin, transferrin, laminin, fibronectin, vitronectin, and the like.

[0101] In some embodiments, the polysaccharide and its derivatives include but are not limited to chitin, chitosan, and the like.

[0102] In some embodiments, the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.

[0103] In some embodiments, the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, etc.;

[0104] In some embodiments, the antioxidants include but are not limited to vitamins such as niacinamide.

[0105] In some embodiments, the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.

[0106] In some embodiments, the metal ions include, but are not limited to, potassium, calcium, magnesium ions, and the like.

[0107] In some embodiments, the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.

[0108] In some embodiments, the positive charge source is urea or nicotinamide mononucleotide.

[0109] In some embodiments, the positive charge source substance is serum, plasma, cell culture medium, animal and plant tissue fluid, animal tissue, etc., or a mixture containing the above positive charge source substances.

[0110] The cell culture medium includes but is not limited to: complete cell culture medium, animal-free cell culture medium, animal protein-free cell culture medium, and chemically defined cell culture medium.

[0111] The present invention unexpectedly discovered that the self-assembling peptide having the structure described in the present invention can be triggered and self-assembled to form a nano-network structure under physiological conditions, cell storage and culture environments, and under conditions of a wide range of endogenous substances in the human body, and can also provide a self-assembling peptide solution system that can exert supporting and repair functions in a solution state.

[0112] Therefore, in a second aspect, the present invention provides a method for initiating the self-assembling peptide of the first aspect to form a three-dimensional network scaffold structure, the method comprising the step of initiating under conditions of a positive charge source.

[0113] In some embodiments, the method includes the step of initiating at a pH of less than 6.

[0114] When the pH of the self-assembling peptide solution is less than 6, it will self-initiate to form a hydrogel.

[0115] In some embodiments, the method includes the step of initiating with a positive charge source at a pH between 6-10.

[0116] In some embodiments, the method comprises the step of initiating with a positive charge source at pH 6-8.0, preferably pH 6.5-7.5, preferably pH 7.0-7.5, more preferably pH 7.2-7.4.

[0117] The solution self-assembling peptide can be dissolved in a neutral or alkaline solvent, and the solution can be adjusted after dissolution. The solvent includes one or more aqueous solutions of sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonia water, etc. that can provide an alkaline environment.

[0118] The solvent can be used to dissolve the self-assembling peptides of the present invention as long as it can provide a neutral or alkaline environmental solution, preferably a physiologically acceptable solution. The pH of the mixed solution of the proteinaceous substance and the self-assembling peptide is adjusted to 6-10, preferably to 6.0-8.0, more preferably 6.5-7.5, and most preferably 7.0-7.5.

[0119] A convenient and safe method for preparing a broad-spectrum, responsive, self-assembling peptide hydrogel under physiological conditions is suitable for use in laboratories, hospitals, and even the field. It is safe, highly operable, simple, and quick, requiring no adjustment of the system's pH, temperature, light, salt, or ion composition. The self-assembling peptide hydrogel of the present invention is formed in situ within half an hour, demonstrating strong operability. Gelation is initiated by endogenous substances, such as tissue fluid, complete cell culture medium, serum-free cell culture medium, and other common substances in the biomedical field, as well as some synthetic drugs. This reduces the requirements of the self-assembling peptide gelation process for application scenarios such as cell culture and storage, tissue filling and repair, and better ensures the biocompatibility of the self-assembling peptide hydrogel of the present invention.

[0120] In addition, because the initiating substances of the present invention are of many types, they can be applied to a variety of application scenarios, such as the application of broad-spectrum responsive peptide hydrogel materials in 3D cell culture and storage, tissue engineering, regenerative medicine, drug delivery, etc., with broad application prospects. For example, the self-assembling peptide hydrogel of the present invention is used to culture different types of cells, realize three-dimensional culture in vitro, and establish a cell model; the self-assembling peptide hydrogel of the present invention loaded with cells / organoids / organs is injected into the animal body for in vivo 3D culture and tissue repair research; or the self-assembling peptide or peptoid solution of the present invention is directly injected with the tissue fluid of the animal / human body to form a peptide hydrogel for use in wound dressings, hemostatic materials, etc.; or after the drug / functional molecule is mixed with the self-assembling peptide or peptoid solution of the present invention to form a peptide hydrogel, it is injected or applied to the wound or lesion in vitro or in vivo as a carrier for sustained release of drugs or functional factors; or the self-assembling peptide hydrogel of the present invention is mixed with a cell suspension for cell preservation. Therefore, the self-assembling peptide hydrogel of the present invention has a wide range of applications and is safe, and does not introduce additional risk substances to the application scenario.

[0121] The advantages of the present invention are also reflected in that the self-assembling peptide of the present invention can be formulated into a polypeptide solution under neutral conditions, and such a polypeptide solution has a low viscosity before the addition of the initiator because it maintains a weak self-assembly morphology, thereby maintaining the convenience of handling the self-assembling peptide solution before use. The self-assembling peptide of the present invention comprises a relatively hydrophobic sequence segment and a relatively hydrophilic sequence segment, and the relatively hydrophilic sequence segment contains at least two consecutive sequence segments that can form a β-turn structure, wherein at least one of the sequence ends of the β-turn structure is an acidic amino acid. Its self-assembly mechanism provides self-assembly power for the interaction between the hydrophobic effect and the β-turn structure, promoting gelation. The acidic amino acids on the relatively hydrophilic sequence segment realize the positively charged substance responsiveness of the polypeptide. The reason is that the hydrophilic domain on the polypeptide contains at least one acidic amino acid, which is negatively charged under neutral conditions. There is a charge repulsion between the polypeptide molecules, and it is impossible to form a tight molecular stack. Even if the hydrophobic effect of the hydrophobic domain provides power for the aggregation of molecules, the electrostatic repulsion between the molecules hinders a stable and orderly arrangement. In addition, the acidic amino acids in the hydrophilic domain are hydrophilic amino acids, which tend to be exposed in the solution. The β-turn structure reduces interference from other side chains near the acidic amino acids. Therefore, the β-turn structure is conducive to the function of acidic amino acids. The β-turn structure makes the side chain groups of the acidic amino acids on the polypeptide more active, exacerbating the repulsion between molecules, affecting the arrangement of molecules, and further increasing the difficulty of self-assembling peptides to form a three-dimensional network scaffold structure. Moreover, when the self-assembling peptide molecules approach each other during movement, they are given acceleration due to electrostatic effects, which increases the kinetic energy of the entire system. Therefore, in a solution containing only polypeptides under neutral conditions, the distribution of the self-assembling peptide molecules is relatively chaotic, and a three-dimensional network scaffold material cannot be formed, so the entire system is in a solution state.

[0122] Once a positive charge source initiator is added to such a self-assembling peptide solution, when positive ions / groups are present, the carbonyl groups on the self-assembling peptides will form electrostatic interactions with them. The negative charges on the self-assembling peptide molecules are "shielded" or "neutralized", the electrostatic repulsion between molecules is reduced, and the β-turn structure in the polypeptide sequence enhances the chimeric interaction between peptides, while threonine, serine and hydroxyproline containing hydroxyl groups will further enhance the interaction between polypeptides due to the formation of hydrogen bonds, and then, driven by hydrophobic interactions and hydrogen bonds, the molecules are orderly aggregated and regularly distributed to form a three-dimensional mesh scaffold material. This triggering mode of the polypeptide of the present invention is particularly advantageous for the preparation of injectable therapeutic hydrogels. It is very convenient for mixing functional components and is also very easy to operate during injection. After being injected into the tissue, it quickly self-assembles to form a gel to exert its effect. When performing 3D cell culture, the polypeptide solution in solution is also very convenient for cell inoculation. After mixing with cells, self-assembly is initiated, thereby achieving the support function for the cells.

[0123] The three-dimensional mesh scaffold material is a nanostructure.

[0124] The initiator is a positive charge source substance, and the positive charge source substance includes a substance with a positively charged group or a positively charged ion.

[0125] In some embodiments, the positive charge source substance is a biomolecule, drug, functional molecule, or small molecule such as metal ions, amino acids, etc., whose number of hydrogen bond acceptors is smaller than that of hydrogen bond donors, or a mixture comprising one or more of the above substances.

[0126] In some embodiments, the biomolecules include but are not limited to organic acids, proteins, polysaccharides and derivatives thereof, and the organic acids include but are not limited to lactic acid, tannic acid, citric acid and the like.

[0127] In some embodiments, the protein is selected from proteins that are capable of donating hydrogen ions under neutral physiological conditions.

[0128] Preferably, the proteins are independently selected from proteins having an isoelectric point (PI) value lower than 7.0 (preferably 3.4-6.05).

[0129] In some embodiments, the protein includes, but is not limited to, fibrinogen, globulin, hemoglobin, transferrin, laminin, fibronectin, vitronectin, and the like.

[0130] In some embodiments, the polysaccharide and its derivatives include but are not limited to chitin, chitosan, and the like.

[0131] In some embodiments, the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.

[0132] In some embodiments, the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, etc.;

[0133] In some embodiments, the antioxidants include but are not limited to vitamins such as niacinamide.

[0134] In some embodiments, the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.

[0135] In some embodiments, the metal ions include, but are not limited to, potassium, calcium, magnesium ions, and the like.

[0136] In some embodiments, the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.

[0137] In some embodiments, the positive charge source is urea or nicotinamide mononucleotide.

[0138] In some embodiments, the positive charge source substance is serum, plasma, cell culture medium, animal or plant tissue fluid, or a mixture containing the above positive charge source substances.

[0139] The cell culture medium includes but is not limited to: complete cell culture medium, animal-free cell culture medium, animal protein-free cell culture medium, and chemically defined cell culture medium.

[0140] It should be noted that if the hydrophobic domain is too hydrophobic, it will easily form an association, while if the hydrophobic domain is too weak, it will not be able to achieve self-assembly. The hydrophilic domain interacts with the initiator to trigger the self-assembly of the self-assembling peptide aqueous solution into a peptide hydrogel. Therefore, the selection of hydrophobic amino acids in the hydrophobic domain, the selection of hydrophilic amino acids in the hydrophilic domain, and the balance and precise combination of hydrophobic and hydrophilic amino acids are particularly important for the self-assembling peptides of the present invention to not form a hydrogel under neutral conditions and to form a hydrogel under initiation conditions.

[0141] The present invention unexpectedly discovered that self-assembling peptides with the structures described herein can be triggered to self-assemble into nanonetwork structures under physiological conditions, in the presence of a wide range of endogenous substances in cell culture environments and the human body. In particular, the use of endogenous physiological substances, such as serum, plasma, cell culture media, plant and animal tissue fluids, and animal tissues, as initiators significantly reduces safety risks in clinical applications, which is particularly important for drug delivery, tissue repair, and regenerative medicine.

[0142] In some embodiments, the three-dimensional mesh scaffold material is in the form of a hydrogel or a dry form of a hydrogel, such as a freeze-dried powder of a hydrogel.

[0143] In a third aspect, the present invention provides a three-dimensional network scaffold material in the form of a hydrogel, wherein the three-dimensional network scaffold material comprises the self-assembling peptide of the first aspect.

[0144] In some embodiments, the three-dimensional mesh scaffold material is obtained by the method of the second aspect.

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

[0146] In some embodiments, the three-dimensional mesh scaffold material is in the form of an injectable hydrogel.

[0147] In some embodiments, the three-dimensional mesh scaffold material is a nanostructure.

[0148] The three-dimensional network scaffold material has more β-sheet structures than the self-assembling peptide.

[0149] In a fourth aspect, the present invention provides a composition comprising the self-assembling peptide of the first aspect and an initiator.

[0150] The composition is in the form of being combined together, or in the form of being combined, wherein the latter is that the self-assembling peptide and the initiator are placed in different containers respectively.

[0151] The initiator is a positive charge source substance, and the positive charge source substance includes a substance with a positively charged group or a positively charged ion.

[0152] In some embodiments, the positive charge source substance is a biomacromolecule, drug, functional molecule, or small molecule such as metal ions, amino acids, etc., in which the number of hydrogen bond acceptors is smaller than the number of hydrogen bond donors, or a mixture containing one or more endogenous or exogenous substances among the above substances.

[0153] In some embodiments, the biomacromolecules include but are not limited to organic acids, proteins, polysaccharides and derivatives thereof, and the organic acids include but are not limited to lactic acid, tannic acid, citric acid and the like.

[0154] In some embodiments, the biomacromolecules include but are not limited to organic acids, proteins, polysaccharides and derivatives thereof, and the organic acids include but are not limited to lactic acid, tannic acid, citric acid and the like.

[0155] In some embodiments, the protein is selected from proteins that are capable of donating hydrogen ions under neutral physiological conditions.

[0156] Preferably, the proteins are independently selected from proteins having an isoelectric point (PI) value lower than 7.0 (preferably 3.4-6.05).

[0157] In some embodiments, the protein includes, but is not limited to, fibrinogen, globulin, hemoglobin, transferrin, laminin, fibronectin, vitronectin, and the like.

[0158] In some embodiments, the polysaccharide and its derivatives include but are not limited to chitin, chitosan, and the like.

[0159] In some embodiments, the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.

[0160] In some embodiments, the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, and the like.

[0161] In some embodiments, the antioxidants include but are not limited to vitamins such as niacinamide.

[0162] In some embodiments, the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.

[0163] In some embodiments, the metal ions include, but are not limited to, potassium, calcium, magnesium ions, and the like.

[0164] In some embodiments, the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.

[0165] In some embodiments, the positive charge source is urea or nicotinamide mononucleotide.

[0166] In some embodiments, the positive charge source substance is serum, plasma, cell culture medium, animal or plant tissue fluid, or a mixture containing the above positive charge source substances.

[0167] The cell culture medium includes but is not limited to: complete cell culture medium, animal-free cell culture medium, animal protein-free cell culture medium, and chemically defined cell culture medium.

[0168] The present invention unexpectedly discovered that under the conditions of multiple initiators, the three-dimensional network scaffold structure formed is more stable. Therefore, such self-assembling peptides are particularly suitable for in vivo applications and have unparalleled advantages over other self-assembling peptides in the prior art. The three-dimensional network scaffold material is a nanostructure.

[0169] In a fifth aspect, the present invention provides the use of the self-assembling peptide of the first aspect, the method of the second aspect, the three-dimensional mesh scaffold material of the third aspect, and the kit of the fourth aspect in one or more selected from the following: regenerative medicine and tissue regeneration; 2D and 3D cell culture and storage; dispersion and embedding filling of microspheres; drug delivery; wound healing; implantable materials; gene therapy; stem cell therapy; and medical cosmetology. DETAILED DESCRIPTION

[0170] The present invention obtains a self-assembling peptide by cleverly controlling the secondary structure of the polypeptide, uniquely selecting hydrophobic amino acids in the hydrophobic domain, uniquely selecting hydrophilic amino acids in the hydrophilic domain, and balancing and precisely coordinating the hydrophobic and hydrophilic amino acids.

[0171] The self-assembling peptide responds to common positive ion / group source substances in the body or common positive ion / group source substances in cell culture medium under neutral conditions of human physiological conditions to form a three-dimensional nanofiber network structure containing the self-assembling peptide.

[0172] The self-assembling peptides of the present invention are in a neutral liquid state during use under human physiological conditions, thus avoiding the risks caused by adjusting pH or introducing other exogenous substances, such as certain metal salt ions, specific proteins, etc.

[0173] The self-assembling peptides of the present invention have a stronger support effect due to the presence of hydroxyproline (O) in the β-turn motif of the hydrophilic domain. In addition, the present invention has found that the presence of hydroxyproline (O) compared to proline (P) in the β-turn motif of the hydrophilic domain, the support effect of the self-assembling peptide is stronger.

[0174] The self-assembling peptide can self-initiate to form a hydrogel in a solution when the pH value is less than 6.

[0175] When the pH of the self-assembling peptide is between 6 and 10, a positive charge source substance is added as an initiator to form a hydrogel.

[0176] The interaction between the acidic amino acids of the self-assembling peptides of the present invention and the initiator triggers the self-assembly of the peptide aqueous solution into a peptide hydrogel. Under neutral conditions, the addition of the initiator produces a positively charged substance, which neutralizes the negatively charged acid ions on the polypeptide molecules, thereby reducing the repulsive forces between the polypeptide molecules. The polypeptide molecules then self-assemble through hydrophobic interactions and hydrogen bonds, ultimately forming a nanoscale three-dimensional network structure.

[0177] The advantages of the present invention are also reflected in that the self-assembling peptides of the present invention can be formulated into a polypeptide solution under neutral conditions, and such a polypeptide solution does not form a three-dimensional mesh scaffold material before the addition of an initiator, thereby maintaining the ease of handling of the self-assembling peptide solution before use. The reason is that the hydrophilic domain on the polypeptide contains at least one acidic amino acid, which is negatively charged under neutral conditions. There is a charge repulsion between the polypeptide molecules, which cannot form a tight molecular stack. Even though the hydrophobic effect of the hydrophobic domain provides the driving force for molecular aggregation, the electrostatic repulsion between the molecules hinders the stable and orderly arrangement. In addition, the acidic amino acids in the hydrophilic domain are hydrophilic amino acids, which tend to be exposed to the solution. The β-turn structure reduces interference from other side chains near the acidic amino acids. Therefore, the β-turn structure is conducive to the functioning of the acidic amino acids. The β-turn structure makes the side chain groups of the acidic amino acids on the polypeptide more active, exacerbating the repulsion between molecules, affecting the arrangement of molecules, and further increasing the difficulty of the self-assembling peptide to form a three-dimensional mesh scaffold structure. Moreover, when the self-assembling peptide molecules approach each other during movement, they are given acceleration due to electrostatic effects, which increases the kinetic energy of the entire system. Therefore, in a solution containing only polypeptides under neutral conditions, the distribution of self-assembling peptide molecules is relatively chaotic and cannot form a three-dimensional network scaffold material.

[0178] Once a positive charge source initiator is added to such a self-assembling peptide solution, when positive ions / groups are present, the carbonyl groups on the self-assembling peptides will form electrostatic interactions with them. The negative charges on the self-assembling peptide molecules are "shielded" or "neutralized", the electrostatic repulsion between molecules is reduced, and the β-turn structure in the polypeptide sequence enhances the chimeric interaction between peptides, while threonine, serine and hydroxyproline containing hydroxyl groups will further enhance the interaction between polypeptides due to the formation of hydrogen bonds, and then, driven by hydrophobic interactions and hydrogen bonds, the molecules are orderly aggregated and regularly distributed to form a three-dimensional mesh scaffold material. This triggering mode of the polypeptide of the present invention is particularly advantageous for the preparation of injectable therapeutic hydrogels. It is very convenient for mixing functional components and is also very easy to operate during injection. After being injected into the tissue, it quickly self-assembles to form a gel to exert its effect. When performing 3D cell culture, the polypeptide solution in solution is also very convenient for cell inoculation. After mixing with cells, self-assembly is initiated, thereby achieving the support function for the cells.

[0179] In particular, under physiological conditions, such as neutral physiological conditions, the addition of protein substances or a mixed system containing protein substances provides positive charges, thereby neutralizing the negatively charged acid ions on the self-assembling peptide molecules, thereby reducing the repulsive force between the self-assembling peptide molecules. The self-assembling peptide molecules then achieve self-assembly through hydrophobic interactions and hydrogen bonds, ultimately forming a three-dimensional network nanostructure.

[0180] Therefore, the advantages of the present invention are also reflected in that the self-assembling peptide of the present invention can be formulated into a self-assembling peptide solution under neutral conditions, and such a self-assembling peptide solution does not form a three-dimensional mesh scaffold material before adding a proteinaceous substance or a mixed system containing a proteinaceous substance, thereby maintaining the stability of the self-assembling peptide solution before use. The reason is that the hydrophilic domain on the self-assembling peptide contains at least one acidic amino acid, which is negatively charged under neutral conditions. There is a charge repulsion between the self-assembling peptide molecules, and it is impossible to form a tight molecular stack. Even if the hydrophobic effect of the hydrophobic domain provides power for the aggregation of molecules, the electrostatic repulsion between molecules hinders the stable and orderly arrangement. In addition, the acidic amino acids in the hydrophilic domain are hydrophilic amino acids, which tend to be exposed in the solution. The structure of the β-turn reduces the interference of other side chains near the acidic amino acids. Therefore, the structure of the β-turn is conducive to the acidic amino acids to play a role. The β-turn structure makes the side chain groups of the acidic amino acids on the self-assembling peptide more active, aggravates the repulsion between molecules, affects the arrangement of molecules, and further increases the difficulty of the self-assembling peptide to form a three-dimensional mesh scaffold structure. Furthermore, when self-assembling peptide molecules approach each other during movement, they are accelerated by electrostatic interactions, increasing the kinetic energy of the entire system. Consequently, in a neutral solution containing only self-assembling peptides, the distribution of the molecules is relatively chaotic, preventing them from forming a three-dimensional mesh scaffold. This advantage is particularly evident in the preparation of injectable hydrogels for therapeutic applications.

[0181] Once a positively charged protein source or a mixed system containing a protein source is added to such a self-assembling peptide solution, the carbonyl groups on the self-assembling peptides will form electrostatic interactions with the positively charged ions / groups. The negative charges on the self-assembling peptide molecules are "shielded," reducing the electrostatic repulsion between the molecules. Hydrophobic interactions and hydrogen bonds lead to orderly aggregation and regular distribution of the molecules, forming a three-dimensional network scaffold material.

[0182] Before the addition of the initiator, the self-assembling peptides formed a network of curved, interwoven nanofibers with uneven fiber distribution, curvature, and strong flexibility. In contrast, the presence of the initiator reduced the curvature of the fibers, significantly increasing their length and diameter. The fiber arrangement also changed, with the fibers now tightly and orderly arranged into bundles that intertwined with each other. Circular dichroism (CD) analysis revealed that after the addition of the initiator, the secondary structure of the nanofibers assembled from the self-assembling peptides was primarily β-turns. β-sheets were added to the secondary structure of the nanofibers, providing a favorable foundation for the formation of more β-sheet structures and a more regular and orderly fiber structure. Furthermore, the secondary structure of the fibers became more diverse, and the increase in secondary structure promoted the stability of the fiber structure.

[0183] The present invention also demonstrates that the introduction of hydroxyproline into the hydrophilic domain significantly enhances the stability of the β-sheet formed by polypeptide self-assembly through an enhanced hydrogen bond network between polypeptides, thereby improving the stability of the self-assembling scaffold and thus enhancing the cell support function. Simultaneously, the β-sheet structure within the hydrophilic region is enhanced by replacing glycine with alanine and aspartic acid with glutamic acid, enhancing the hydrophobicity of the amino acid side chains without destroying the β-turn structure in the hydrophilic region. This can also increase the stability of the self-assembling material through hydrophobic interactions, thereby making the scaffold material more supportive of cells.

[0184] In addition, since the cell membrane of red blood cells is negatively charged as a whole, in the absence of positive ions / groups, self-assembling peptides cannot form an effective three-dimensional network scaffold material and cannot provide conditions to support cells. In the present invention, under the stimulation of different types of positive ions / groups, self-assembling peptides form a three-dimensional network scaffold material, allowing red blood cells to achieve three-dimensional distribution.

[0185] The cell proliferation rate of cells grown in a two-dimensional environment shows a gradually decreasing trend after a period of culture, the cell state deteriorates, and then the total cell number drops sharply; the three-dimensional mesh scaffold formed by self-assembly of the self-assembling peptides of the present invention enables cells to grow at a relatively uniform and stable growth rate throughout the entire culture cycle, indicating that the three-dimensional mesh scaffold material of the present invention is not only non-toxic to cells and has good biocompatibility, but also enables cells to proliferate stably in a good state for a long time under the support of the mesh scaffold.

[0186] Adding the self-assembling peptide of the present invention to a cell culture medium containing natural substances that are positive charge sources can highly restore the growth environment of cells in vivo and achieve long-term three-dimensional cell culture in vitro.

[0187] The storage effect of the hematopoietic stem cell storage system using the three-dimensional mesh scaffold formed by self-assembly of the self-assembling peptide of the present invention is better than that of the traditional two-dimensional storage, and the cell survival rate is greatly improved.

[0188] Glossary:

[0189] Hydrogel is a hydrophilic polymer material that can form a three-dimensional network structure through chemical or physical crosslinking. Hydrogel materials can come from natural and synthetic sources. Natural polymers such as chitosan, alginate, hyaluronic acid (HA), collagen, gelatin, etc. have the advantages of being biodegradable and carrying integrin binding sites, but they are immunogenic. Synthetic polymers such as polyethylene glycol (PEG), polyacrylamide (PAM), polyvinyl alcohol (PVA) and polymethyl methacrylate (PMMA) have the advantages of strong mechanical properties, customizability, and low immunogenicity, but lack inherent biological functions and must undergo important post-processing to induce the desired response in vivo.

[0190] Hydrogels have the following properties:

[0191] 1. Good biocompatibility: The polymer contains a large number of hydrophilic groups, which can absorb dozens of times more water than its own amount, and has the characteristics of swelling but not dissolving in water, and has good water retention capacity;

[0192] 2. Similar to the extracellular matrix: Through structural design, the physical, chemical and mechanical properties of the hydrogel can be made similar to those of the extracellular matrix, which is conducive to cell growth and reproduction;

[0193] 3. Biodegradability: Some natural polymer materials are biodegradable, preventing secondary damage caused by implant removal. It is these unique advantages that make hydrogels shine in biomedical materials.

[0194] In the present invention, when a peptide having a structure having both a hydrophilic surface and a hydrophobic surface self-assembles under the conditions described in the present invention, especially physiological conditions, a hydrogel is obtained by encapsulating water.

[0195] Peptide Self Assembly is a short chain of amino acids with alternating charge and polarity domains. When dissolved in a neutral solvent and physiological salt concentration, these peptides spontaneously assemble into hierarchical nanostructures through hydrogen bonds, ionic bonds, hydrophobic interactions, or van der Waals forces. Materials derived from these assemblies have the advantages of being non-toxic, non-immunogenic, non-thrombogenic, degradable, and easily metabolized. At the same time, nanofibers have the same size scale as natural ECM fibers and can be easily designed to mimic the stiffness of various soft tissues. They can also be further functionalized by attaching cell-interacting peptide domains or cytokines and growth factors. Therefore, they can be used to design biologically relevant culture environments and improve the control of proliferating cell populations.

[0196] Self-assembling peptides can be used as drug carriers in hydrogels for wound treatment and as peptide nanofibers for cancer treatment, enabling the sustained release of small molecules, growth factors, and monoclonal antibodies. For example, self-assembling peptides can be used to promote angiogenesis within regenerating tissues and to repair skin wounds using peptide-based scaffolds. However, the application of self-assembling peptides in injectable therapeutic hydrogels is still in its infancy.

[0197] The alternating hydrophilic and hydrophobic amino acid residues in the self-assembling peptides allow them to retain large amounts of water and thus form hydrogels. The side chains of the hydrophilic residues can directly interact with water, with water molecules forming inclusion complexes to surround the side chains of the hydrophobic residues. The number of hydrophobic and hydrophilic residues in the self-assembling peptides requires clever design and proportioning. If there are too many hydrophobic residues, the self-assembling peptide will be insoluble in water and precipitate out of the water; on the other hand, if there are too many hydrophilic residues, the self-assembling peptide will be highly water-soluble and therefore unable to form a hydrogel. In addition, it is also necessary to precisely and cleverly induce the peptide molecules to self-assemble into ordered nanostructured materials, such as nanofibers, nanotubes, and nanovesicles.

[0198] "Amino acid" includes those naturally occurring as well as non-naturally occurring amino acids, such as D-natural amino acids, beta and gamma derivatives. According to standard terminology, amino acid residue sequences can be named using three-letter or one-letter codes, for example: alanine (Ala, A); arginine (Arg, R); asparagine (Asp, N); aspartic acid (Aspartic acid, D), cysteine ​​(Cysteine, C); glutamine (Glutamine, Q); glutamic acid (Glutamic acid, E); glycine (Gly, G); histidine (His, H), isoleucine (Ile, I); leucine (Leu, L), lysine (Lysine, L), methionine (Met, M); phenylalanine (Ala, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W), tyrosine (Tyr, Y); valine (Val, V); selenocysteine ​​(Sec, U); hydroxyproline (Hyp, O).

[0199] In the present invention, a "peptide" is an amino acid chain. In particular, a peptide is 2 to 40 amino acids in length.

[0200] In the present invention, "self-assembly" refers to the aggregation of polypeptides into an ordered structure under normal environmental conditions.

[0201] In the present invention, "β-fold" refers to a relatively extended periodic folded zigzag main chain conformation in a polypeptide chain, which is arranged in parallel or antiparallel, thereby forming a β-fold (sheet). The parallel or antiparallel conformation is determined based on the direction of the peptide arrangement from N to C terminus. Parallel arrangement means that the peptide chains are arranged from N to C terminus. Antiparallel arrangement means that the peptide chains are arranged in opposite directions (i.e., the first peptide chain is arranged from N to C terminus, and the opposite second peptide chain is arranged from C to N terminus). The parallel arrangement may include the two ends of the peptide being staggered with each other due to peptide translation. At least half of the length of the peptide is involved in the interaction force between peptides. In the antiparallel arrangement, the polypeptides are usually arranged in a line to provide two flush endpoints. This is a typical end-to-end complementary peptide.

[0202] A β-turn is an irregular secondary structure in proteins that causes a change in the direction of the polypeptide chain. β-turns often occur at the corner where the peptide chain makes a 180° turn. A β-turn consists of 3-5 amino acid residues, with the second residue being either proline (P) or hydroxyproline (O). A β-turn motif generally refers to a turn structure stabilized by hydrogen bonds between the carbonyl oxygen atom of the nth amino acid residue and the amide proton of the n+3th amino acid residue. β-turns, also known as β-elbows, reverse elbows, or β-loops, serve to connect β-strands.

[0203] The term "hydrophobicity" used in the present invention refers to a property of being inclined to repel water or being completely insoluble in water.

[0204] The term "hydrophilicity" in the present invention refers to the property of easily absorbing water and having a strong polar group that easily interacts with water.

[0205] Hydrophilic amino acids, also known as polar amino acids, have polar R groups that can generally form hydrogen bonds with water molecules, thus having a certain affinity for water molecules. Hydrophilic amino acids include: S, T, Y, C, U, N, Q, D, E, O, R, K, and H.

[0206] Hydrophobic amino acids, also known as non-polar amino acids, have non-polar R groups and have low or no affinity for water molecules, but have a high affinity for fat-soluble substances. They include: G, A, V, L, I, P, M, F, and W.

[0207] "Nanostructure" refers to structures with nanometer dimensions. Nanostructures can be any one-dimensional, two-dimensional, or three-dimensional shape, including nanofilms, nanofibers, nanorods, nanowires, nanofiber networks, nanospheres, nanohelices, and mixtures thereof. A nanostructure's surface has a one-dimensional structure at the nanoscale, meaning the surface thickness of the object is between 0.1 nm and 100 nm. Nanotubes have two nanometer dimensions, with diameters ranging from 0.1 to 100 nm and lengths potentially exceeding. Spherical nanoparticles have three nanometer dimensions, meaning the particle's size in each spatial dimension is between 0.1 and 100 nm.

[0208] When the protein solution is at a certain pH, the protein has an equal tendency to dissociate into positive and negative ions, that is, it becomes a zwitterionic ion with a net charge of zero. The pH of the solution at this time is called the isoelectric point (pI) of the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0209] Figures 1A-1B show that when arginine is used as an initiator, the self-assembling peptide without the addition of an initiator is in a liquid state (A). When arginine is present, the self-assembling peptide is a hydrogel (B). Figure 1C shows that when tissue fluid is used as an initiator, the self-assembling peptide can form a hydrogel, which remains in a gel state after being squeezed out of a syringe.

[0210] 2A-2J show that different initiating substances mixed with self-assembling peptides can all produce the three-dimensional network scaffold material of the present invention.

[0211] FIG3 shows a hydrogel three-dimensional mesh scaffold material formed using fibrinogen, transferrin, and γ-globulin as initiators.

[0212] 4A-4B show the fluorescence changes of thioflavin T in peptide fiber hydrogels formed using fibrinogen, transferrin, and γ-globulin as initiators.

[0213] FIG5 shows the effects of different sequence structures of self-assembling peptides on the support of red blood cells in forming a three-dimensional mesh scaffold.

[0214] FIG6 shows the effect of the triggering component on the support of the self-assembling peptide solution on red blood cells.

[0215] Figure 7 shows the changes in rheological properties of different self-assembling peptide sequence structures, in order: SEQ ID NO: 7 (A), SEQ ID NO: 3 (B), SEQ ID NO: 2 (C), SEQ ID NO: 1 (D), SEQ ID NO: 5 (E), SEQ ID NO: 6 (F), SEQ ID NO: 4 (G), SEQ ID NO: 10 (H), SEQ ID NO: 8 (I).

[0216] Figures 8A-8E show confocal laser scanning electron microscopy images of the self-assembling peptide material of the present invention that supports cells after being primed by spermine (B), high-glucose complete medium (C), tissue fluid (D), and serum (E), respectively. The concentration of the self-assembling peptide is 0.1 wt.%, and (A) is a buffer solution without any priming component.

[0217] Figures 9A-9B show the relative viability (A) and growth curve (B) of cells cultured on a three-dimensional network scaffold material induced to form using different concentrations of the self-assembling peptides of the present invention in complete culture medium over 5 days;

[0218] Figures 10A-10D show confocal laser scanning electron microscopy images of cells cultured for 3 days (A) and 7 days (C) after the self-assembling peptide material of the present invention was added to the cell culture system to induce its assembly into a fibrous network scaffold, wherein (B) and (D) represent typical cell clusters in the 3-day and 7-day culture systems, respectively. The concentration of the self-assembling peptide material used in each experimental group was 0.1 wt.%;

[0219] Figures 11A-11D show the addition of 0.1 wt.% of the self-assembling peptide material of the present invention to a cell culture system to induce assembly into a fiber network scaffold and culture cells. The diameter of the cell spheres was measured on the fifth, tenth, and fifteenth days (A), and characteristic cell spheres in the culture system were photographed (B, C, D);

[0220] FIG12 shows that 0.1 wt.% of the self-assembling peptide material of the present invention was added to the cell culture system to induce the assembly of the self-assembling peptide material into a fiber network scaffold and the cells were cultured. After 15 days, the cells were harvested and their relative viability was measured over 5 days.

[0221] FIG13 shows the effects of not (2D) and using (3D) the self-assembling peptide scaffold solution on the cell survival of mouse mesenchymal stem cells during refrigerated storage.

[0222] Figures 14A-14C show that the self-assembling peptide solution can stably disperse L-polylactic acid microspheres, where A shows that the microspheres precipitate in the aqueous solution, but are evenly suspended and dispersed after mixing with the self-assembling peptide solution; B shows that the protein-responsive self-assembling peptide mixture of the microspheres is liquid; C shows that after the protein-responsive self-assembling peptide mixture of the microspheres obtained in Figure 14B is mixed with tissue fluid, the protein-responsive self-assembling peptide / L-polylactic acid microsphere solution quickly forms a hydrogel.

[0223] Figures 15A-15C show that the protein-responsive self-assembling peptide solution can stably disperse polycaprolactone: A microspheres precipitate in the aqueous solution, but are evenly suspended and dispersed after mixing with the self-assembling peptide solution; B shows that the protein-responsive self-assembling peptide mixture of the microspheres is liquid; C shows that after the protein-responsive self-assembling peptide mixture of the microspheres obtained in B is mixed with tissue fluid, the protein-responsive self-assembling peptide / L-polylactic acid microsphere solution quickly forms a hydrogel.

[0224] Example

[0225] Example 1 Synthesis of self-assembling peptide compounds

[0226] The self-assembling peptides or peptide-like compounds of the present invention are synthesized by a standard solid-phase self-assembling peptide synthesis method, and the amino acid sequences are shown in SEQ ID NOs: 1-32.

[0227] Example 2 Preparation of Polar Peptide or Peptoid Solution

[0228] The self-assembling peptide or peptoid of Example 1 is added to water or phosphate buffer (PBS) buffer (pH = 7.2-7.4, unless otherwise specified, the PBS buffer used in this description is of this pH value), and alkali solution is added dropwise until the self-assembling peptide is completely dissolved. The solution is adjusted to a neutral pH of 7.2 to obtain a 5 wt.% self-assembling peptide or peptoid mother solution, which is sterilized by autoclaving and stored at 4°C for future use. The mother solution is used to adjust the concentration of the self-assembling peptide to obtain a self-assembling peptide or peptoid material of a predetermined concentration. A certain amount of the self-assembling peptide or peptoid mother solution is diluted with PBS buffer to obtain a 0.5 wt.% self-assembling peptide or peptoid solution.

[0229] Example 3 Preparation of three-dimensional network scaffold materials formed by self-assembling peptides under the stimulation of positive ions / groups

[0230] The self-assembling peptide or peptoid solution obtained in Example 2 is evenly mixed with the initiating substance solution, and the concentration ratio of the self-assembling peptide or peptoid to the initiating substance in the mixed solution is ensured to be in the range of (1-100):(1-100), thereby obtaining a self-assembling, three-dimensional network scaffold with multiple initiating substances. The pH value of the obtained mixed solution is neutral (about pH 6 to about pH 8, preferably about pH 6.5-7.5, preferably about pH 7-7.5).

[0231] Taking the self-assembling peptide represented by the amino acid sequence IIIIIGSIIGPGGDGPGGV (SEQ ID NO: 1) as an example, the initiating substance is arginine. A 0.5 wt.% peptide solution (see Figure 1A) is in liquid form. When the sample bottle is inverted, the solution flows back and collects at the bottle mouth. In the presence of arginine, a peptide hydrogel with a peptide concentration of 0.5 wt.% self-assembles in response to the initiating substance arginine (see Figure 1B). When the sample bottle is inverted, the hydrogel is formed, preventing the material from falling. Figure 1C shows that using tissue fluid as the initiator, the self-assembling peptide can form a hydrogel, which remains in a gel state after being extruded from a syringe. This also demonstrates similar or equivalent effects on peptide hydrogel materials formed in response to other types of initiating substances.

[0232] Taking the self-assembling peptide represented by the amino acid sequence LLLLLGSVLGPAGEGPAGE (SEQ ID NO: 29) as an example, a 2% concentration of the self-assembling peptide quickly forms a hydrogel after being mixed with an equal volume of human tissue fluid. The hydrogel can be drawn up with a syringe and remains in a gel state after being extruded by injection. It also has the same or similar effects on peptide hydrogel materials formed in response to other types of triggering substances.

[0233] According to this method, a self-assembling peptide hydrogel material with any predetermined peptide concentration ratio of self-assembling peptide or peptoid to initiating substance described in the present invention can be prepared. The results are shown in Table 1 below.

[0234] Example 4 Structural Characterization of the Scaffold Material Formed by the Self-Assembling Peptides of the Present Invention

[0235] Experiment 1 Transmission Electron Microscopy (TEM)

[0236] The polar peptide or peptoid is exemplified by SEQ ID NO: 3 (IIIIIGSIIGOGGEGPGGV); the initiating substance is exemplified by polylysine, spermine, gentamicin, lysine, arginine, spermidine, kanamycin, chitosan, and magnesium ions.

[0237] Experimental method: The mother liquor of the self-assembling peptide material obtained in Example 3 (SEQ ID NO: 3) and the three-dimensional mesh scaffold material formed by self-assembly in response to the initiating substance obtained in Example 2 were diluted with ultrapure water. In order to obtain the nanoscale morphology of the hydrogel under physiological conditions, they were incubated at 37°C for 1 hour, 10 μL was taken and placed on a 300-mesh carbon support film copper grid (Xinxing Bairui), and vacuum dried. The sample was stained with a 2wt.% phosphotungstic acid negative staining solution for 60 seconds each time, repeated three times. The stained copper grid was placed at room temperature to dry. Imaging observation was performed using a Talos G2 200X transmission electron microscope.

[0238] Experimental results: As shown in Figures 2A-2J, different initiating substances can be mixed with self-assembling peptides to obtain the three-dimensional mesh scaffold material of the present invention. The initiating substances corresponding to AJ are: polylysine, spermine, gentamicin, lysine, arginine, spermidine, kanamycin, chitosan, and magnesium ions. The structure formed by the self-assembling peptide material (Figure 2A) is composed of curved and interwoven nanofibers. The fibers are unevenly distributed, curved, and highly flexible. The presence of the initiating substance reduces the curvature of the fibers (Figures 2B-2J), significantly increases the fiber length and diameter, and changes the arrangement of the fibers. The fibers are tightly and orderly arranged to form fiber bundles, which are interwoven with each other.

[0239] Experiment 2: Circular Dichroism (CD) Analysis

[0240] Circular dichroism spectroscopy is the most widely used method for determining protein secondary structure and monitoring conformational changes of protein molecules induced by external conditions. This method is used to detect liquids, and the results obtained are closer to the secondary structure of proteins in real physiological environments. It is a fast, simple and relatively accurate method for studying protein conformation.

[0241] Experimental method: Preparation of self-assembling peptide or derivative solution for CD detection: The obtained 0.2 wt % self-assembling peptide solution was diluted with PBS buffer to a self-assembling peptide concentration of 0.02 wt %.

[0242] Preparation of protein solution for CD detection: Laminin, fibronectin, fibrinogen, transferrin, γ-globulin, and vitronectin were added to ultrapure water to a protein concentration of 0.02 wt%.

[0243] Preparation of self-assembling peptide or derivative samples: The 0.2 wt % self-assembling peptide solution obtained in Example 2 was diluted with PBS buffer to a self-assembling peptide concentration of 0.01 wt %, and incubated at 37° C. for one hour.

[0244] To prepare the peptide hydrogel samples of the present invention, a 0.02 wt% self-assembling peptide solution was mixed with a 0.02 wt% protein solution at a 1:1 volume ratio. The mixture was incubated at 37°C for one hour to obtain a final self-assembling peptide concentration of 0.01 wt%. Because the self-assembling peptide / protein hydrogel is a swelling system, although the self-assembling peptide concentration in this experiment was lower than 0.1 wt%, the results still confirmed the interaction between the self-assembling peptide and the protein.

[0245] 400 μL of each prepared sample was placed in a rectangular quartz cell with a 1 mm optical path length. Circular dichroism detection was performed in the 190 nm to 260 nm wavelength range using a MOS-450 / AF-CD spectrometer (Bio-Logic, Claix, France) at room temperature with a resolution of 0.5 nm and a scan speed of 0.5 nm / s. Each set of measurements was performed in triplicate after background subtraction. The results are shown in Figure 3 for peptide hydrogels formed with the self-assembling peptide of SEQ ID NO: 13 (IIIIIGOGIIGPGGEGPGGE) and fibrinogen, transferrin, and gamma globulin, respectively.

[0246] The results of circular dichroism detection show that the responsiveness of the self-assembling peptide or its derivatives to proteins is manifested as a change in secondary structure. Figure 3 shows that when the self-assembling peptide or its derivatives exist alone (see control group) in a neutral environment, the secondary structure of the nanofibers assembled therein is mainly β-turns. In the system where proteins are introduced separately, it can be seen that the positive peak near 200nm in the circular dichroism spectrum shifts to varying degrees. This shows that compared to when the self-assembling peptide or its derivatives exist alone and are not triggered, the response to protein increases the secondary structure of the nanofibers assembled by the self-assembling peptide or its derivatives, providing a favorable basis for forming more β-fold structures and making the fiber structure more regular and orderly. In addition, a negative peak appears near 192nm when the protein group is added (see fibrinogen triggering group, transferrin triggering group, and gamma globulin triggering group). This is the embodiment of the characteristic polymeric structure formed by the participation of proline or hydroxyproline in the self-assembling peptide in the assembly of the self-assembling peptide in the fiber secondary structure. This shows that the response to these three substances makes the secondary structure of the fiber assembled by the self-assembling peptide of the present invention more diverse, and the increase in secondary structure promotes the stability of the fiber structure. This experiment shows that although self-assembling peptides or their derivatives can assemble into nanofibers with specific secondary structures by themselves, their responsiveness to proteins enables them to assemble into nanofibers with more complex and ordered structures, thus macroscopically exhibiting a three-dimensional network structure with tight cross-linking and uniform pores as shown in Experiment 1.

[0247] Experimental Conclusion: Experiment 2 demonstrated that the presence of the initiating substance caused the polar peptides to self-assemble into a gel-like network, which is beneficial for cell adhesion and three-dimensional culture. Combining Experiments 1 and 2, it was confirmed that the interaction between the self-assembling peptides and the initiating substance altered the self-assembly pathway, promoting the formation of a three-dimensional network scaffold material with a distinct microscopic morphology compared to that observed without the initiating substance.

[0248] When the self-assembling peptide or peptoid of the present invention is used, or when the self-assembling peptide structure of the present invention is met, similar experimental results to those of Experiments 1 and 2 can be obtained under the stimulation of the triggering substance, and are not listed in detail one by one.

[0249] Experiment 3: Thioflavin T fluorescence experiment

[0250] Experimental Methods: Thioflavin T can bind to the β-sheet structure of self-assembling peptides, thereby enhancing fluorescence intensity. In this experiment, we used SEQ ID NO:13 (IIIIIGOGIIGPGGEGPGGE) to monitor the fluorescence changes of Thioflavin T and verify the secondary structural changes of self-assembling peptides during self-assembly. 800μM (0.148 wt%), 400μM (0.072 wt%), and 200μM (0.036 wt%) peptide sols were mixed with 0.072 wt% of a trigger in a 1:1 volume ratio. Ultrapure water was used instead of the trigger for the control. After 15 minutes of stabilization, the sols were mixed with an equal volume of a 100μM Thioflavin T solution. Fluorescence emission was measured in the 450-550 nm range, with five spectra collected for each sample. The excitation wavelength was 442 nm, and the excitation and emission slits were 5 nm and 2.5 nm, respectively.

[0251] Experimental Results: CD spectral data were validated using thioflavin T (ThT), which can stain peptide fibers rich in β-sheet structure, confirming the presence of β-sheets and revealing their relationship with peptide concentration. β-sheets were present in the self-assembling peptide sol, and their content was positively correlated with the self-assembling peptide concentration, indicating that the self-assembling peptide is concentration-dependent (Figure 4A). This is because the increase in peptide monomers facilitates intermolecular contact, thereby increasing the degree of assembly. After adding the initiator, the β-sheet content of the gel-generated group was much higher than that of the sol group (Figure 4B). CD and ThT staining data indicate that under neutral pH conditions, the supramolecular polymers generated by protein initiator-activated self-assembling peptides exhibit a typical β-sheet structure.

[0252] Example 5 Effects of Amino Acid Sequence Structure and Initiating Components on the Physical and Functional Properties of the Peptide Self-Assembly Material of the Present Invention, and Verification of the Self-Assembly Mechanism

[0253] Experiment 1: Verification of red blood cell support by different sequences under liquid conditions

[0254] In this experiment, taking red blood cells as an example, we verified the supporting effect of amino acid sequences on cells, especially the supporting effect of hydroxyproline and alanine in the amino acid sequence on cells.

[0255] Experimental method: SEQ ID NO: 1 (IIIIIGSIIGPGGDGPGGV), SEQ ID NO: 2 (IIIIIGSIIGPGGEGPGGV), SEQ ID NO: 3 (IIIIIIGSIIGOGGEGPGGV), and SEQ ID NO: 7 (IIIIIIGSIIGOGAEGPGGV) self-assembling peptide stock solutions were prepared with PBS buffer at a concentration of 0.1 wt%, and sterilized at high temperature.

[0256] Use 2 ml transparent glass bottles, and configure 2 ml of self-assembled peptide solutions with 0.1 wt%, 0.05 wt%, and 0.01 wt% of the four materials respectively (containing fibronectin with a final concentration of 0.1 wt%), and a blank group (without self-assembled peptide solution, containing 0.1 wt% of fibronectin). Add 5×10 9 erythrocyte mother liquor to make the final concentration 1×10 8 , and pipette evenly. Take pictures every 4 hours to compare the supporting effects of different materials on erythrocytes.

[0257] Experimental conclusion: Among self-assembled peptides with similar amino acid sequences, hydroxyproline and alanine have a significant impact on the cell-supporting ability of the scaffold solution under liquid conditions. As shown in Figure 5, under high-concentration conditions (0.1 wt%, 0.05 wt%), all four self-assembled peptide solutions can effectively support erythrocytes with a concentration of 1×10 8 . However, under low-concentration conditions (0.01 wt%), hydroxyproline, alanine, and glutamic acid in the hydrophilic domain show a significant impact on the erythrocyte-supporting ability. The four self-assembled peptide solutions show obvious sedimentation at 8 hours, 24 hours, 32 hours, and 48 hours respectively. The supporting abilities of the four self-assembled peptides from weak to strong are: SEQ ID NO:1 (IIIIIGSIIGPGGDGPGGV) < SEQ ID NO:2 (IIIIIGSIIGPGGEGPGGV) < SEQ ID NO:3 (IIIIIGSIIGOGGEGPGGV) < SEQ ID NO: 7 (IIIIIGSIIGOGAEGPGGV). Based on the scaffold network formed by the fibronectin response, compared with SEQ ID NO:1, glutamic acid in the hydrophilic region of SEQ ID NO:2, glutamic acid and hydroxyproline in the hydrophilic region of SEQ ID NO:3, and glutamic acid, hydroxyproline, and alanine in the hydrophilic region of SEQ ID NO:7 successively show an increasingly stronger enhancing effect on the cell-supporting ability. Hydroxyproline is abundantly present in the collagen tissue of animals and can enhance the elasticity and support force of the protein matrix through the hydrogen bonds formed by its hydroxyl groups. In this patent, by introducing hydroxyproline, the stability of the β-sheet formed by the self-assembly of self-assembled peptides can be significantly enhanced through the enhanced hydrogen bond network between self-assembled peptides, thereby improving the stability of the self-assembled scaffold and enhancing the cell-supporting function. At the same time, the β-sheet structure in the hydrophilic region can enhance the hydrophobicity of the amino acid side chains through the substitution of single alanine for glycine and the substitution of glutamic acid for aspartic acid without destroying the β-turn structure in the hydrophilic region, and can also increase the stability of the self-assembled material through hydrophobic interactions, making the scaffold material more supportive of cells.

[0258] Experiment 2: Red blood cell support experiment, the effects of different triggering components

[0259] Experimental method: Take 0.16mL of red blood cell stock solution (9.17×10 9 / mL), diluted with 10mM PBS to 10mL (9.17×10 7 / mL), take 4.5mL of red blood cell dilution solution and add it to a 10mL round-bottom test tube, and add 4.5mL of 4mmol / L Calcein-AM solution, incubate at 37℃ for 20min, centrifuge at 4℃, 1500r / min, centrifuge for 3min, remove the supernatant, add PBS to resuspend, repeat 5 times to obtain about 1.5mL of washed red blood cell resuspension (1×10 8 / mL) and set aside.

[0260] Experimental results: PBS buffer was added to the red blood cell resuspension to dilute the red blood cells to 1×10 7 / mL, mix well and let stand for 4 hours. The results are shown in tube A in Figure 6.

[0261] The 1 wt% self-assembling peptide SEQ ID NO: 3 solution obtained in Example 2 was added to make the final peptide concentration in the system 0.05 wt% and the final red blood cell concentration 1×10 7 / mL, mix well and let stand for 4 hours. The results are shown in tube B in Figure 6.

[0262] A mixed solution of polypeptide SEQ ID NO: 3 obtained in Example 3 and lysine was added to the red blood cell resuspension, i.e., a three-dimensional network scaffold material formed by the polypeptide in the presence of lysine, to achieve a final peptide concentration of 0.05 wt.% in the system. The mixture was mixed and allowed to stand. The result is shown in tube C in FIG6 .

[0263] The self-assembling peptide SEQ ID NO: 3 obtained in Example 2 and a mixed solution of gentamicin were added to the erythrocyte resuspension, i.e., a three-dimensional network scaffold material formed by the peptide in the presence of gentamicin, so that the final peptide concentration in the system was 0.05 wt.%, mixed and allowed to stand. The result is shown in tube D in FIG6 .

[0264] The self-assembling peptide SEQ ID NO: 3 obtained in Example 2 and a mixed solution of polylysine were added to the red blood cell resuspension, i.e., a three-dimensional network scaffold material formed by the self-assembling peptide in the presence of polylysine, so that the final peptide concentration in the system was 0.05 wt.%, mixed and allowed to stand. The result is shown in tube E in Figure 6.

[0265] The self-assembling peptide SEQ ID NO: 3 obtained in Example 3 and a mixed solution of fibrinogen were added to the red blood cell resuspension, i.e., a three-dimensional network scaffold material formed by the self-assembling peptide in the presence of fibrinogen, so that the final peptide concentration in the system was 0.05 wt.%, mixed and allowed to stand. The result is shown in tube F in Figure 6.

[0266] The results of the control group without adding self-assembling peptides and positive ion / group substances are shown in tube A in Figure 6. The red blood cells are concentrated at the bottom of the centrifuge tube. In tube B, the red blood cells undergo obvious sedimentation, with a trend of three-dimensional distribution, but it is not obvious. In tube CE, the red blood cells present a three-dimensional distribution, and the height increase of the red part can be observed in the centrifuge tube. Compared with tube CF, under the stimulation of different types of positive ions / groups, the self-assembling peptides form a three-dimensional network scaffold material, allowing the red blood cells to achieve a three-dimensional distribution, while the system is still in a solution state. Combined with the results of experiment 1, it can be seen that the response of the self-assembling peptide to positive ions / groups is reflected in the change of the secondary structure formed by molecular assembly. This change causes the self-assembling peptide to form fibers of different morphologies, and the distribution pattern of the fibers changes (experiment 1). This microscopic change explains the difference between tube CF and tubes A and B in this experiment. The red blood cells are adsorbed on the three-dimensional nanofibers formed in response to the self-assembling peptides, and are able to present a three-dimensional distribution. Only self-assembling peptides (B tubes) cannot form effective three-dimensional network scaffold materials and provide conditions for supporting cells because the cell membrane of red blood cells is negatively charged as a whole and there is a lack of positive ions / groups.

[0267] Experiment 3: Rheological experiment, the influence of the secondary structure and amino acid sequence of self-assembling peptides

[0268] Experimental method: Preparation of self-assembling peptide material samples: Use the 2 wt% self-assembling peptide solution obtained in Example 2, SEQ ID NO: 1 (IIIIIGSIIGPGGDGPGGV), SEQ ID NO: 2 (IIIIIGSIIGPGGEGPGGV), SEQ ID NO: 3 (IIIIIIGSIIGOGGEGPGGV), SEQ ID NO: 4 (IIIIGSIIGOGGEGPGGV), SEQ ID NO: 5 (IIIIIGSIIGOGGEGPGGGV), SEQ ID NO: 6 (IIIIIGSIIGOGGEGPGGV), SEQ ID NO: 7 (IIIIIGSIIGOGAEGPGGV), SEQ ID NO: 8 (IIIIIGSIIOGGAEGPGGV), SEQ ID NO: 9 (IIIIIIGSIIGOGGVGPGGV), SEQ ID NO: 10 (IIIIIIGSIIGOGAEGPGGVGPGGV).

[0269] Preparation of hydrogel samples formed by self-assembling peptide solution in response to complete cell culture medium: dilute the 2 wt% self-assembling peptide material stock solution obtained in Example 2 with phosphate buffer, add calf serum, and the final concentration of self-assembling peptide is 0.5%, and the final concentration of serum is 10 v / v%, and the pH is neutral.

[0270] The storage modulus (G') of the mixed solution was measured using a MARS 60 rheometer on a 20 mm plate. To determine the rate of 3D nanomatrix formation, the solution was placed on the plate immediately after preparation. A 500 μm gap was used, with mineral oil added to prevent sample dehydration, and data collection began. A dynamic time sweep (DTS) experiment was performed to monitor the change in storage modulus (G') over time (1 Hz frequency, 1% strain) for 2000 minutes.

[0271] Experimental results: The mechanical strength of the peptide hydrogel of the present invention can effectively provide support and encapsulation effects for cells, functional molecules, drugs, etc., which is conducive to the realization of three-dimensional cell culture, tissue repair, drug sustained release, etc. Experiments have shown that by mixing the peptide solution and serum, self-assembling peptides can be immediately obtained to respond to the triggering substance and assemble into a hydrogel material with a storage modulus greater than 10. The operation is convenient and the gelation time is short. Comparing different sequences, it was found (see Figures 7A-7I) that SEQ ID NO: 7 (IIIIIGSIIGOGAEGPGGV) > SEQ ID NO: 3 (IIIIIGSIIGOGGEGPGV) > SEQ ID NO: 2 (IIIIIIGSIIGPGGEGPGGV) ≥ SEQ ID NO: 1 (IIIIIIGSIIGPGGDGPGGV); this experimental phenomenon is consistent with the results of Experiment 3, that is, hydroxyproline can enhance the mechanical strength of the hydrogel formed by the present invention, thereby enhancing the supporting function of the scaffold material. At the same time, the results of comparing SEQ ID NO: 5 (IIIIIGSIIGOGGEGPGGGV) with SEQ ID NO: 6 (IIIIIGSIIGOGGEGPGV) showed that when the number of β-turn amino acids was 6 (SEQ ID NO: 5) and 3 (SEQ ID NO: 6), and the β-turn structure was increased to 3 consecutive (SEQ ID NO: 10), or the number of hydrophobic amino acids was 4 (SEQ ID NO: 4), the self-assembling peptide could still form a hydrogel structure with weak elasticity. In Figure 7, A corresponds to the sequence SEQ ID NO: 7, B corresponds to the sequence SEQ ID NO: 3, C corresponds to the sequence SEQ ID NO: 2, D corresponds to the sequence SEQ ID NO: 1, E corresponds to the sequence SEQ ID NO: 5, F corresponds to the sequence SEQ ID NO: 6, G corresponds to the sequence SEQ ID NO: 4, H corresponds to the sequence SEQ ID NO: 10, and I corresponds to the sequence SEQ ID NO: 8.

[0272] In SEQ ID NO:8, we completely destroyed the formation conditions of the first β-turn in SEQ ID NO:7 by adjusting the 10th-11th amino acid GO in SEQ ID NO:7 to OG, forming the sequence SEQ ID NO:8; As a result, we found that SEQ ID NO:8 completely lost its self-assembly response to serum. This phenomenon further proves that two consecutive β-turns are the most basic conditions for the self-assembly ability of the self-assembling peptide in the present invention. By comparing SEQ ID NO:9 (IIIIIGSIIGOGGVGPGGV) with SEQ ID NO:4 (IIIIGSIIGOGGEGPGGV), we found that when the end of the β-turn is no longer connected to an acidic amino acid, the self-assembling peptide aqueous solution no longer responds to serum, and it can form a hydrogel in a neutral aqueous solution; here we further verified that the acidic amino acid connected to the end of the β-turn in the hydrophilic region of at least one self-assembling peptide is a prerequisite for the self-assembling peptide self-assembly material of the present invention to respond to the triggering component.

[0273] Example 6 Application of the peptide hydrogel material of the present invention in the field of biomedicine

[0274] Experiment 1: Cell support effect of the self-assembling peptide of the present invention in three-dimensional space after initiation

[0275] Growing cells on a two-dimensional surface is an artificial and unnatural culture method because it is very different from the internal environment of the organism where cells can grow optimally. Therefore, cells harvested by traditional 2D cell culture methods grow in an environment that is very different from the inside of the organism, resulting in loss of cell structure and stimulus response functions. Three-dimensional cell culture can better simulate the natural environment in which cells survive in the body, and can highly maintain the normal biochemical reactions and cell-to-cell interactions of the cells themselves. Therefore, cells in a three-dimensional environment have characteristic biological signals that can affect their functions such as migration, adhesion, proliferation, and gene expression, and their responses to endogenous and exogenous stimuli are closer to their reactions in vivo. Specific cellular processes in tissue engineering, such as differentiation, have been shown to occur more easily in a three-dimensional environment than in a two-dimensional environment. Achieving three-dimensional cell culture requires that the materials used to culture cells have excellent cell support effects and biocompatibility.

[0276] This experiment used the common adherent cell HepG2 as a cell model, using SEQ ID NO: 13 (IIIIIGOGIIGPGGEGPGGE), serum as a representative of a mixture of natural biomacromolecules carrying positively charged groups, and spermine as a representative of positively charged natural small molecules to trigger self-assembling peptides, and explored the effect of the three-dimensional mesh scaffolds formed by their assembly to support cells. At the same time, in order to explore the cell-supporting effect of the self-assembling peptide material of the present invention when applied in real cell culture and biomedicine, high-glucose culture medium, tissue fluid, and serum were used as substances carrying positive charge sources to trigger the self-assembling peptides of the present invention to simulate the actual use environment.

[0277] HepG2 cells were cultured in a cell culture flask at 37°C in a mild and humid environment with a CO2 content of 5%. After culture, the cells were collected and resuspended in an appropriate amount of high-glucose medium (serum content of 10%) to obtain a concentration of 5×10 5 Cell suspension of cells / mL. The prepared cell suspension and Calcein-AM / PI dye solution were mixed evenly in a volume ratio of 2: 1, and incubated in the dark at 37°C for 15 minutes. The self-assembling peptide hydrogel material of the present invention, which was assembled into a three-dimensional network scaffold by each positively charged substance, was added to a glass-bottomed culture dish (the method for configuring the self-assembling peptide hydrogel is shown in Example 3), and then the cell suspension after incubation in the dark was added and mixed until the total volume in the glass-bottomed culture dish was 200 and the final concentration of the self-assembling peptide was 0.1wt%. When preparing the control group samples, an equal volume of PBS buffer was used to replace the self-assembling peptide hydrogel material of the present invention in the experimental group. Each experimental sample was then observed using an LSM 980with Airyscan2 fast super-resolution laser confocal microscope.

[0278] Figure 8 shows the results of this experiment. In the control group without the self-assembling peptide hydrogel of the present invention, as shown in Figure 8A, the cells naturally sank to the bottom of the glass-bottom culture dish, were not supported, and were distributed in a 2D plane. When the self-assembling peptide of the present invention triggered by a positively charged source substance was introduced into the system, as shown in Figures 8B, 8C, 8D, and 8E, the cells in the system were supported by the three-dimensional mesh scaffold formed by the self-assembling peptides, presenting a three-dimensional distribution in a 3D environment.

[0279] This experimental phenomenon proves that the self-assembling peptide material of the present invention can be triggered by the positively charged natural small molecule spermine to form a 3D network scaffold with good cell support effect (Figure 8B), which can effectively realize the growth and culture of cells in three-dimensional space in vitro, preventing them from sinking and adhering to the wall and then growing in a two-dimensional environment. Complete cell culture medium (Figure 8C), tissue fluid (Figure 8D) and serum (Figure 8E) are comprehensive systems that are widely used in the fields of cell culture and biomedicine and carry a large amount of positively charged source substances. Figures 8C, 8D, and 8E show that the polypeptide materials of the present invention can be triggered by these three comprehensive systems to assemble into three-dimensional network scaffolds and show good cell support effects. The entire initiation process is mild and does not require the addition of new substances other than the above three comprehensive systems. It is effective, convenient, and harmless to cells, showing the excellent huge potential of the polypeptide materials of the present invention in the fields of cell culture and biomedicine.

[0280] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0281] Experiment 2: Cytocompatibility of the Self-Assembling Peptide Material of the Present Invention

[0282] In order to prove that the self-assembling peptide material of the present invention has good biocompatibility, this experiment took SEQ ID NO: 13 (IIIIIGOGIIGPGGEGPGGE) as an example to measure the relative viability and proliferation number of HepG2 cells for five days.

[0283] The concentration prepared in Experiment 1 was 5×10 5 The concentration of 5×10 cells / mL cell suspension was prepared by 4 cells / mL of cell suspension. The concentration obtained is 5×10 4 Cells were seeded at a density of 10 cells / mL into a 96-well plate, so that each well contained approximately 5,000 cells. The experimental group was added with a self-assembling peptide sol prepared according to the method described in Example 2, so that the concentration of the peptide in the final system was 0.1%, 0.3%, 0.5%, and 0.7%, respectively. The control group was added with an equal volume of PBS buffer to replace the peptide solution. After mixing, each group was subjected to a 5-day experiment. During this period, no additional culture medium was replaced or added. During the experiment, 10% of the culture volume of the CCK-8 kit was added every 24 hours. After incubation for 30 minutes, the OD value of each group was measured using a microplate reader (parameter setting: main wavelength 450nm, secondary wavelength 630nm). All experiments were set up in 5 parallel groups. The experimental results are shown in Figure 9A. The relative cell viability was calculated using the following formula:

[0284] Relative cell viability (%) = (A t -A 0t ) / (A1-A 01 )×100%;

[0285] Among them, A t A is the average OD value measured after culturing cells for t days. 0t is the average OD value of the blank background measured after t days, A1 is the average OD value measured after culturing cells for 1 day, and A 01 It is the average OD value of the blank background measured one day later.

[0286] The concentration obtained was 5×10 4 Cells were seeded at a density of 10 cells / mL in a 12-well plate, with each well containing approximately 50,000 cells. The experimental groups were supplemented with a self-assembling peptide sol prepared according to the method described in Example 2, resulting in peptide concentrations of 0.1%, 0.3%, 0.5%, and 0.7%, respectively. An equal volume of PBS buffer was added to the control group to replace the peptide solution. All groups were mixed and then subjected to a 5-day experiment without additional culture medium changes or additions. Cells were collected and counted every 24 hours during the experiment. All experiments were performed in five parallel groups. The results are shown in Figure 9B.

[0287] The results of the relative cell viability experiment measured by the CCK-8 test kit (Figure 9A) show that during the 5-day experimental process, the cells in each experimental group with the addition of the self-assembling peptide material of the present invention showed a uniform and continuous steady growth in cell viability, and the cells were in good condition, showing the typical characteristics of adherent cells growing in three-dimensional space, indicating that the cells have been supported by the three-dimensional mesh scaffold formed by the polypeptide assembly; while the cell viability in the control group first surged and then dropped sharply, showing the typical characteristics of adherent cells growing in two-dimensional space. Two-dimensional culture will lead to its proliferation inhibition due to limited growth space and contact inhibition of adherent cells, and a large number of cells will die in the later stage of culture. These two phenomena illustrate that the self-assembling peptide of the present invention not only has good biocompatibility and no toxic effects on cells, but also can realize long-term culture of cells in three-dimensional space in vitro, expand cell growth space in the same culture volume, and restore the growth state of cells in vivo.

[0288] The cell growth curve over 5 days (Figure 9B) shows that the cells grown in a two-dimensional environment (control group) had a higher total cell count than all experimental groups for the first four days, but the cell proliferation rate showed a gradual downward trend, the cell state deteriorated, and the total cell count dropped sharply on the fifth day. In contrast, all experimental groups grew at a relatively uniform and stable growth rate throughout the culture period, indicating that the polypeptide material of the present invention is not only non-toxic to cells and has good biocompatibility, but also enables cells to proliferate stably and in a good state for a long time under the support of the mesh scaffold assembled from this material. This experimental phenomenon also confirms the conclusions of the relative cell viability measurement experiment.

[0289] And by summarizing the above two experimental phenomena, it can be concluded that, when comparing each experimental group, the cell activity and proliferation rate will show slight differences as the concentration of the polypeptide material is adjusted, indicating that in practical applications, the cell proliferation rate can be adjusted by adjusting the concentration of the material used to better meet personal use needs.

[0290] Experiment 2 used HepG2 cells as a cell model, which not only confirmed the conclusion of Experiment 1 that "the polypeptide material of the present invention can effectively support cells in vitro", but also proved that the material of the present invention has good biocompatibility, and that during the culture period, the cells can maintain a high activity and continue to proliferate in a good state and at a uniform rate.

[0291] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0292] Experiment 3: Cell culture using the self-assembling peptide hydrogel of the present invention

[0293] The inherent property of adherent cells is that they are easy to aggregate and form spheres under conditions that prevent them from adhering to a certain plane, that is, they are easy to grow into cell spheres in a three-dimensional space. There are concentration gradients of oxygen, nutrients, and metabolic waste in cell spheres, which can simulate the various characteristics of solid tissues. It is an important 3D physiological model for studying the occurrence of solid tumors and stem cell differentiation, and is widely used in the field of biomedicine. Moreover, cell spheres are simpler than other 3D physiological models. During the research process, imaging analysis can be achieved through common experimental means, such as optical, fluorescence, and confocal microscopy, which simplifies the experimental process in the research. In this experiment, the common adherent cell HepG2 was cultured with the polypeptide material of the present invention induced by high-glucose culture medium, and the formation of cell spheres was used to verify that the material of the present invention can achieve cell culture similar to that in organisms in vitro.

[0294] Prepare the concentration of 5×10 5 The concentration of 1×10 cells / mL cell suspension was prepared by 5 Taking SEQ ID NO: 13 (IIIIIGOGIIGPGGEGPGGE) as an example, the concentration obtained is 1×10 5 A cell suspension of 100 cells / mL was seeded into a 24-well plate containing the self-assembling peptide material of the present invention (final peptide concentration per well: 0.1 wt.%), resulting in approximately 100,000 cells per well. The culture medium was replaced twice weekly using the "half-volume medium exchange method" during the culture period. To more intuitively observe changes in cell morphology and distribution during the culture period, cells were stained with Calcein-AM / PI solution after three and seven days of culture and observed under an LSM 980 with Airyscan2 fast super-resolution laser confocal microscope. The results are shown in Figure 10.

[0295] As shown in Figure 10A, incubation with only 0.1 wt.% of the polypeptide material of the present invention, which was activated by high-glucose medium to assemble into a three-dimensional mesh scaffold, not only allowed HepG2 cells to form spheres, but also did so in just 72 hours. This demonstrates that the self-assembling peptide material of the present invention can achieve in vitro cell culture similar to in vivo cell growth and effectively promotes the rapid sphere formation of adherent cells. After 72 hours, the spheres were observed to be of similar size, with cells in good condition and no apoptosis (Figure 10B). After 7 days of culture, the diameter of the spheres increased significantly (Figure 10C), demonstrating that cells can proliferate in a three-dimensional environment within this culture system. Specifically, the addition of the polypeptide of the present invention to a cell culture medium containing a positively charged natural substance can highly replicate the in vivo cell growth environment. Observation of the spheres after 7 days of culture (Figure 10D) revealed that the spheres were of similar size and cells in good condition. The occurrence of a small number of apoptotic cells occurs because, as cells continue to proliferate in the spheres, the concentration gradient of oxygen, nutrients, and metabolic waste gradually increases. Cells in the center of the spheres undergo apoptosis due to decreasing oxygen and nutrients and increasing metabolic waste, which is a natural phenomenon.

[0296] We also observed and recorded the diameters of 50 cell spheres in the culture system under an inverted microscope after 5, 10, and 15 days of culture. The cell sphere diameter data collected over these three days were averaged and plotted into a bar graph (Figure 11D). This test examined whether HepG2 cells cultured in the polypeptide material of the present invention could achieve long-term in vitro proliferation, and photographed characteristic cell spheres (Figures 11A, 11B, and 11C). As shown in Figure 12, in a culture system containing 0.1 wt.% of the primed polypeptide material of the present invention, HepG2 cells were supported and subsequently grew and proliferated in the form of cell spheres. As the culture time increased, the cell spheres became increasingly compact and their diameters increased at a similar rate, indicating that HepG2 cells could stably proliferate at a uniform rate over a 15-day culture period, indicating that the material can be used for long-term in vitro three-dimensional cell culture. The relatively uniform cell proliferation rate also indicates that the cells were in good condition during the culture period, and that the material of the present invention did not damage the cells.

[0297] Both Experiments 1 and 2 demonstrated that the polypeptide material of the present invention possessed excellent cell-supporting properties. Experiment 2 also demonstrated its excellent biocompatibility and suitability for cell culture. Experiment 3 demonstrated that the addition of the polypeptide of the present invention to a cell culture medium containing a natural substance that provides a positive charge source could highly replicate the in vivo cell growth environment, enabling long-term three-dimensional cell culture in vitro.

[0298] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0299] Experiment 4: Evaluation of the status of cells harvested after culture using the self-assembling peptide hydrogel of the present invention

[0300] In this experiment, the cell spheres cultured in Experiment 3 containing 0.1 wt.% of the polypeptide SEQ ID NO: 13 (IIIIIGOGIIGPGGEGPGGE) of the present invention triggered by high-glucose culture medium were centrifuged and digested into single cells. By comparing the cell activity between cells cultured with the material of the present invention and cells not cultured with the material of the present invention, the applicability of the material of the present invention in the fields of cell culture and biomedicine was further evaluated.

[0301] The cell spheres cultured for 15 days in Experiment 3 were collected by centrifugation. The cell spheres were digested into single cells using trypsin and then centrifuged and collected. An appropriate amount of high-glucose medium was added to the collected cells to make the concentrations of 5×10 5 cells / mL and 5×10 4 Cell suspension with a concentration of 5×10 4 Cell suspensions of 5 × 10 cells / mL were inoculated into 96-well plates, with each well containing 5000 cells. 5 The concentration of 5×10 cells / mL cell suspension was prepared by 4 A cell suspension of 5000 cells / mL was seeded into a 96-well plate, with 5,000 cells per well, to serve as the control group. Cell viability was tested over a five-day period, with the culture medium replaced daily. Five parallel groups were set up.

[0302] As shown in Figure 12, cells harvested after culturing with the polypeptide material of the present invention in high-glucose medium showed almost identical trends in cell activity over five days compared to the control group, with the cell activity of the experimental group slightly exceeding that of the control group each day. This indicates that cells can maintain their original physiological activity after culturing with a system containing the self-assembling peptide material of the present invention assembled into a three-dimensional network of fibers. This demonstrates that the material of the present invention does not cause physiological damage to cells during the cell culture process, and can safely and reliably enable cells to proliferate in a state highly similar to in vivo growth while maintaining their original physiological activity, achieving expansion without compromising their physiological functions.

[0303] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0304] Experiment 5: Cell Storage Application

[0305] Experimental Methods: This experiment uses the 4°C storage of mouse mesenchymal stem cells as an example. The isolated mouse mesenchymal stem cells were pipetted evenly and aliquoted into 10 ml cryopreservation tubes. The tubes were pipetted evenly to a cell concentration of approximately 1×106 cells / mL. In the 3D group, mouse serum and 1% double-antibody storage solution in 50% MAP + 50% SFEM were added, and the peptide SEQ ID NO: 20 (FLIVIGOGIIGOGGEGPGGE) was added to a final concentration of 0.05%. In the 2D group, mouse serum and 1% double-antibody storage solution in 50% MAP + 50% SFEM were added, and the tubes were then stored in a 4°C refrigerator.

[0306] Cell viability test:

[0307] (1) Thoroughly mix the stored cells by pipetting, take 500 μL of cell suspension into each 1.5 mL EP tube, add PBS to wash, and centrifuge at 1500 rpm for 10 min;

[0308] (2) Discard the supernatant, resuspend with 150 μL of 1% PBA solution, then add 150 μL of live-dead cell staining working solution, mix thoroughly, incubate at 37°C for 15 min in the dark, and detect using flow cytometry.

[0309] Experimental results: In order to understand the changes in the survival rate of mesenchymal stem cells during the storage period, we used the Calcein-AM / PI live / dead double staining kit to double-stain them and used a flow cytometer to detect them. As shown in Figure 13, starting from the third day of storage, the survival rate of mouse mesenchymal stem cells in the 3D storage group was significantly higher than that in the 2D storage group, with a significant statistical difference (79% vs. 67%). And on the fifth day of storage, it still had a high cell survival rate (71% vs. 43%), and the survival rate increased by nearly 30%. This shows that the storage effect of the mesenchymal stem cell storage system used in the present invention is better than traditional 2D storage, which greatly improves the cell survival rate.

[0310] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0311] Experiment 6: Application of the hydrogel of the present invention as a dispersed filler in medical aesthetics

[0312] Experimental Method: The synthesized self-assembling peptide represented by SEQ ID NO.: 25 (IIIIIGOGIIGOGGEGPGGV) was dissolved in deionized water and vortexed until fully dissolved to obtain a 1.5% self-assembling peptide solution. The pH of the solution was adjusted to 7.4 with 0.1M NaOH solution. Figure 14A shows, from left to right, the peptide solution, the precipitated L-polylactic acid microsphere solution, and a mixture of the two. The peptide solution was autoclaved and then mixed with L-polylactic acid microspheres (particle size between 25-60 μm). Finally, the solution was aliquoted to obtain a 5% microsphere-containing self-assembling peptide mixed injection for injection. The solution became opaque, and the microspheres were evenly and stably distributed in the self-assembling peptide solution (Figure 14B). The resulting microsphere-containing self-assembling peptide mixed injection was placed at room temperature for one month. The obtained injections were uniformly suspended, with no obvious solid-liquid separation. After adding 1 / 5 tissue fluid, hydrogel formation was observed (Figure 14C).

[0313] Similarly, using the same method and steps, SEQ ID NO.: 27 (FLIVIGSIIGOGAEGPGGV) at a concentration of 1.5% can also achieve support for 5% polycaprolactone (PCL) microspheres. As shown in Figure 15A, from left to right are a transparent SEQ ID NO.: 27 polypeptide solution, a precipitated polycaprolactone (PCL) microsphere solution, and a mixture of the two. The high-temperature sterilized polypeptide solution was mixed with polycaprolactone (PCL) microspheres (particle size between 25-50um) to make the content of polycaprolactone (PCL) microspheres 5%. It can be seen that the microspheres are uniformly and stably distributed in the self-assembling peptide solution (Figure 15B). The obtained self-assembling peptide mixed injection containing microspheres was placed at room temperature for one month. It can be seen that the obtained injections are all in a uniform suspension state, with no obvious solid-liquid separation phenomenon. After adding 1 / 5 tissue fluid, a gel formation phenomenon can be seen (Figure 15C).

[0314] In addition, except for SEQ ID NO.: 25 and 27, the self-assembling peptides represented by SEQ ID NO.: 1-7 and 9-32 of the present invention can have the same function on a variety of other medical beauty regeneration microspheres (Table 2).

[0315] Table 2 Common medical aesthetic microspheres:

[0316] Experimental conclusion: This experimental phenomenon proves that the hydrogel material of the present invention, that is, the three-dimensional network scaffold self-assembled by protein-responsive self-assembling peptides in response to proteins, can effectively support medical aesthetic microspheres. It can play a good dispersing and supporting role for the microspheres when it is not activated. After being activated by proteins in the tissue fluid, it can quickly form a hydrogel, thereby playing a local shaping function in the tissue; the entire experimental process does not require the addition of other ingredients, and the operation is convenient.

[0317] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

Claims

1. A self-assembling peptide comprising a hydrophobic domain and a hydrophilic domain, wherein the hydrophilic domain comprises at least two consecutive β-turn regions.

2. The self-assembling peptide according to claim 1, wherein the at least one β-turn region comprises or is linked to one or more acidic amino acids at its terminal end, preferably comprises or is linked to one acidic amino acid.

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

6. The self-assembling peptide according to claim 3, wherein the β-turn motif in at least one β-turn region comprises or is linked to an amino acid selected from glutamic acid (E), valine (V), leucine (L), isoleucine (I), aspartic acid (D) and lysine (K) at the terminal end.

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

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

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

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

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

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

13. The self-assembling peptide according to claim 1, wherein the hydrophobic domain comprises: LLLL (SEQ ID NO.:65), FIIII (SEQ ID NO.:66), IIII (SEQ ID NO.:67), IIII (SEQ ID NO.:68), ILILI (SEQ ID NO.:69), FLFLF (SEQ ID NO.:70), IVIVI (SEQ ID NO.:71), VLFIIV (SEQ ID NO.:72), VLIII (SEQ ID NO.:72) NO.:73), IVALF (SEQ ID NO.:74), LFIVL (SEQ ID NO.:75), FIAIV (SEQ ID NO.:76), FIIIV (SEQ ID NO.:77), Ac-VLFIIV (SEQ ID NO.:78), Ac-IVIVI (SEQ ID NO.:79), Ac-IIIIII (SEQ ID NO.:80), IIIIII (SEQ ID NO.:78) NO.:81), FLIVI (SEQ ID NO.:82), FLIIA (SEQ ID NO.:83), FIFIF (SEQ ID NO.:84), IFIFI (SEQ ID NO.:85), IAILI (SEQ ID NO.:86) or LLLLL (SEQ ID NO.:87).

14. The self-assembling peptide according to claim 1, further comprising a linker domain comprising 2-8 amino acid residues, preferably 4-5 amino acid residues; and The connecting domain comprises small side chain amino acids, amino acids with hydroxyl groups on the side chains and / or hydrophobic amino acids far away from the hydrophobic region, The small side chain amino acids are selected from G, A and S, the amino acids with hydroxyl groups on the side chains are selected from S, T and O, and the hydrophobic amino acids far from the hydrophobic domain are selected from I, V, L, F and A, Preferably, the connecting domain has an amino acid sequence selected from the group consisting of: GPOGI (SEQ ID NO.: 88), GPOGV (SEQ ID NO.: 89), GPOGL (SEQ ID NO.: 90), GSII (SEQ ID NO.: 91), GSGII (SEQ ID NO.: 92), GSVI (SEQ ID NO.: 93), GOII (SEQ ID NO.: 94), OGII (SEQ ID NO.: 95) or GTVI (SEQ ID NO.: 96), wherein, S, T, and O can be interchanged with each other. More preferably, the connecting domain has an amino acid sequence selected from the group consisting of: GSVL (SEQ ID NO.:97), GSII (SEQ ID NO.:91), GTII (SEQ ID NO.:98), GTVI (SEQ ID NO.:96), GOVI (SEQ ID NO.:99), GSVI (SEQ ID NO.:93), GSGII (SEQ ID NO.:92), GSGVI (SEQ ID NO.:100), GOII (SEQ ID NO.:94), OGII (SEQ ID NO.:95), GOGVI (SEQ ID NO.:101) or GOGII (SEQ ID NO.:102).

15. The self-assembling peptide according to claim 1, which has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32.

16. A method for forming a three-dimensional network scaffold material from the self-assembling peptide according to any one of claims 1 to 15, the method comprising the step of inducing the self-assembling peptide to form a three-dimensional network scaffold material under conditions inducing a positive charge source.

17. The method according to claim 16, wherein the initiating self-assembling peptides to form a three-dimensional network scaffold material under the conditions of a positive charge source comprises: Initiation is performed at a pH less than 6; or, Initiation is carried out at a pH of 6-10 by a positive charge source.

18. The method according to claim 17, wherein the positive charge source is a biomacromolecule, a drug, a functional molecule, a metal ion, an amino acid, or a mixture comprising one or more endogenous or exogenous substances thereof. The biomacromolecules are selected from organic acids, proteins, polysaccharides and their derivatives, Preferably, the organic acid is selected from lactic acid, tannic acid and citric acid; Preferably, the protein is a protein that can provide hydrogen ions under neutral physiological conditions. Preferably, the protein has an isoelectric point PI value lower than 7.0, preferably 3.4-6.

05. Preferably, the protein is selected from the group consisting of fibrinogen, globulin, hemoglobin, transferrin, laminin, fibronectin and vitronectin; Preferably, the polysaccharide and its derivatives are selected from chitin and chitosan; The drug is selected from antibiotics and dopamine, Preferably, the antibiotic is selected from kanamycin and gentamicin; The functional molecules are selected from antioxidants and cell proliferation promoting components, Preferably, the antioxidant is a vitamin antioxidant. Preferably, the cell proliferation promoting component is spermine or spermidine; Preferably, the metal ion is potassium ion, calcium ion or magnesium ion; Preferably, the amino acid is lysine, arginine, polylysine, or polyarginine; Preferably, the positive charge source substance is serum, plasma, cell culture medium, animal or plant tissue fluid, or a mixture of one or more thereof, or animal tissue.

19. A scaffold material comprising the self-assembling peptide according to any one of claims 1 to 15, or prepared by the method according to any one of claims 16 to 18.

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

21. A composition comprising the self-assembling peptide according to any one of claims 1 to 15 and the positive charge source according to claim 18.

22. Use of the self-assembling peptide of any one of claims 1-15, the method of any one of claims 16-18, the scaffold material of claim 19 or 20, or the composition of claim 21 in one or more selected from the group consisting of: regenerative medicine and tissue regeneration; 2D and 3D cell culture and storage; dispersion and embedding filling of microspheres; drug delivery; wound healing; implantable materials; gene therapy; stem cell therapy; and medical cosmetology.

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