Protein-responsive self-assembling peptide and application thereof

Abstract

According to the protein-responsive hydrogel and the preparation method thereof, the hydrogel comprises a nanofiber three-dimensional network structure formed by self-assembly of polypeptide or a derivative solution of the polypeptide under induction of protein, in-vitro three-dimensional culture can be achieved, and the hydrogel is used for tissue repair; the material can be used as a wound dressing, a hemostatic material, a material for dispersing regenerated microspheres such as medical poly-L-lactic acid, polycaprolactone and the like, a material for shaping in tissues, a carrier for slowly releasing drugs or functional factors, and a material for preserving cells.

Description

Protein-responsive self-assembling peptides and their applications Technical Field

[0001] The present invention relates to the field of biomedical materials, in particular to protein-responsive 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. The self-assembly process of self-assembling peptides is mainly driven by a series of non-covalent bond forces such as hydrogen bonding, electrostatic interaction, hydrophobic interaction, and π-π stacking between self-assembling peptide molecules and their derivatives. Self-assembling peptides can self-assemble into scaffolds with three-dimensional network structures, enabling the system to complete the transition from solution to gel. Not only can they highly reproduce the natural extracellular matrix (ECM) niche, providing synergistic biochemical and biophysical cues to guide cell proliferation, migration, and differentiation, but they also have the characteristic of being injectable. This has attracted increasing attention in cell culture, tissue engineering, and the entire biomedical field. However, the self-assembly of self-assembling peptides usually requires harsh preparation conditions, and requires certain environmental conditions, exogenous substances, or time limits to initiate, such as: specific temperature (CN106083634A, the initiation temperature must be >40°C), pH (CN109776651A, initiation must be under alkaline conditions), light (CN103992486A, initiation requires ultraviolet light with a wavelength of 300-400nm), or other additional conditions (CN114507270A, ultrasound is required to assist in the construction of a three-dimensional network structure), or the introduction of enzymes (CN110325204A, additional enzymes are required). As an initiator) or other exogenous substances (CN114344481A, dimethyl sulfoxide, which poses a safety risk to the body, needs to be introduced as a solubilizing agent during preparation, or a sufficiently long time needs to be ensured (CN113416264A, the preparation process takes 48 hours) to complete self-assembly. These conditions may require certain equipment, or are not conducive to the survival or safety of cells or animals, or are not conducive to the convenience of operation, thereby limiting the scope of use of hydrogels. Some hydrogels that are self-assembled by endogenous substances (such as substances contained in the scene where the hydrogel is to be used) are often limited in their application scenarios due to the relatively single initiating substance.

[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, especially in the presence of proteins in organisms, and can perform supporting and repair functions in solution will have very important application prospects.

[0004] Summary of the Invention

[0005] The present invention provides a protein-responsive self-assembling peptide, which can respond and self-assemble to form a nano-network structure in the presence of protein, especially endogenous extensive protein conditions, and can also play a supporting and repairing role in the solution state.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0047] In some embodiments, the amino acid sequence of the hydrophobic domain is hydrophobic compared to the amino acid sequence of the hydrophilic domain.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0062] IIIIIGSIIGPGGDGPGGV(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 initiated by a proteinaceous substance or a mixed system containing a proteinaceous substance to form a three-dimensional network scaffold material.

[0094] The protein substances are selected from proteins that can provide hydrogen ions under neutral physiological conditions; preferably, the proteins are independently selected from proteins with an isoelectric point (PI) value lower than 7.0 (preferably 3.4-6.05).

[0095] In some embodiments, the proteinaceous substance is selected from laminin, fibronectin, fibrinogen, globulin, hemoglobin, vitronectin, transferrin, or a combination comprising one or more thereof.

[0096] Mixed systems containing proteinaceous substances include, but are not limited to, complete cell culture medium, serum-free culture medium, animal tissue, cell preservation medium or other cell or drug delivery systems.

[0097] In some embodiments, the mixed system containing protein substances is serum, plasma, cell culture medium, animal or plant tissue fluid, or animal tissue, etc.

[0098] 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 the conditions of protein substances or mixed systems containing protein substances, especially under physiological conditions, cell storage and culture environments and organisms, preferably under the conditions of mixed systems containing protein substances in the human body, and a self-assembling peptide solution system that can exert supporting and repair functions in a solution state.

[0099] The three-dimensional mesh scaffold material has a nanostructure.

[0100] In a second aspect, the present invention provides a method for forming a scaffold material from the protein-responsive self-assembling peptides of the first aspect, the method comprising the step of inducing the self-assembling peptides to form a scaffold material using a proteinaceous substance.

[0101] Preferably, the method comprises the step of mixing the self-assembling peptide into a proteinaceous substance or into a mixed system containing a proteinaceous substance; or the method comprises the step of injecting or implanting the self-assembling peptide into a mixed system containing the proteinaceous substance.

[0102] The proteinaceous substances are selected from proteins that tend to provide hydrogen ions under neutral physiological conditions; preferably, the proteinaceous substances are independently selected from proteins having an isoelectric point (PI) value lower than 7.0 (preferably 3.4-6.05).

[0103] In some embodiments, the proteinaceous substance is selected from laminin, fibronectin, fibrinogen, globulin, hemoglobin, vitronectin, transferrin, or a combination comprising one or more thereof, or a mixed system comprising one or more thereof, wherein the mixed system includes but is not limited to complete cell culture medium, serum-free culture medium, animal tissue, cell preservation medium or other cell or drug delivery system.

[0104] In some embodiments, the mixed system containing protein substances is serum, plasma, cell culture medium, animal and plant tissue fluid, animal tissue, etc.

[0105] In some embodiments, the method includes the step of initiating with a proteinaceous substance or a mixed system comprising a proteinaceous substance at a pH between 6-10.

[0106] In some embodiments, the method comprises the step of initiating with a proteinaceous substance or a mixed system containing a proteinaceous substance at pH 6.5-8.0, preferably pH 7.0-7.5, more preferably pH 7.2-7.4.

[0107] 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.

[0108] 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 environment solution, preferably a physiologically acceptable solution. The pH of the mixed solution of the proteinaceous substance and the self-assembling peptide is adjusted to 6.5-10, preferably 6.0-8.0, more preferably 6.5-7.5, and most preferably 7.0-7.5.

[0109] The protein-responsive self-assembling peptide forms the scaffold material within 30 minutes, preferably within 15 minutes, and more preferably within 10 minutes, at a temperature of 0-90°C, preferably at a temperature of 15-50°C, and a pH value of ≤9, under the conditions of a proteinaceous substance or a mixed system containing a proteinaceous substance.

[0110] The acidic amino acids of the self-assembling peptide of the present invention interact with protein substances or protein substances and polypeptides in a mixed system containing protein substances, triggering the self-assembly of the peptide aqueous solution into a scaffold material in the form of a hydrogel.

[0111] 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.

[0112] 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 completely form a three-dimensional mesh scaffold material before adding a proteinaceous substance or a mixed system containing a proteinaceous substance. Therefore, due to its low viscosity, it is very easy to mix, extract, inject, etc., and has very good operational convenience. 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 a driving force for the aggregation of molecules, the electrostatic repulsion between 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 the interference of other side chains near the acidic amino acids. Therefore, the β-turn structure 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. Moreover, when the self-assembling peptide molecules approach each other during movement, they are given acceleration due to electrostatic interaction, which increases the kinetic energy of the entire system. Therefore, in a solution containing only self-assembling peptides under neutral conditions, the distribution of the self-assembling peptide molecules is relatively chaotic and cannot fully form a three-dimensional network scaffold material.

[0113] Once a positively charged protein source or a mixed system containing protein substances is added to such a self-assembling peptide solution, when the 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", the electrostatic repulsion between the molecules is reduced, and hydrophobic interactions and hydrogen bonds cause the molecules to aggregate and distribute in an orderly manner, forming a three-dimensional network scaffold material, thereby forming a hydrogel on a macro scale.

[0114] The scaffold material is a three-dimensional mesh scaffold material and has a nanostructure.

[0115] It should be noted that if the hydrophobic domain is too hydrophobic, it is easy to form an association, while if the hydrophobic domain is too weak, self-assembly cannot be achieved. The hydrophilic domain interacts with protein substances or mixed systems containing protein substances to trigger the self-assembly of the self-assembling peptide aqueous solution into a 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 coordination of hydrophobic and hydrophilic amino acids are particularly important for the self-assembling peptide of the present invention to not form a hydrogel under neutral conditions and to form a hydrogel under initiation conditions.

[0116] The present invention unexpectedly discovered that self-assembling peptides with the structures described herein can self-assemble into nanonetwork structures under physiological conditions, in a wide range of proteinaceous substances, or mixed systems containing proteinaceous substances, found in cell storage and culture environments and the human body. In particular, using endogenous physiological substances, such as serum, plasma, cell culture media, and animal and plant tissue fluids, as mixed systems containing proteinaceous substances significantly reduces safety risks in clinical applications, which is particularly important for drug delivery, human tissue repair, and regenerative medicine.

[0117] 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.

[0118] In the hydrogel, the concentration of the protein-responsive self-assembling peptide is ≥0.1 wt %, preferably 0.3-4 wt %, and more preferably 0.5-1 wt %.

[0119] The hydrogel has functions such as self-repair. Under the action of mechanical force, the gel state of the hydrogel can be destroyed and then restored to the gel state after the mechanical force disappears. The recovery time does not exceed 10 minutes. After recovery, the storage modulus of the hydrogel is at least 70% of that before destruction, preferably at least 85% of that before destruction, and more preferably more than 95% of that before destruction.

[0120] 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 protein-responsive self-assembling peptide according to the first aspect.

[0121] In some embodiments, the three-dimensional mesh scaffold material is obtained by the method of the second aspect.

[0122] 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.

[0123] In some embodiments, the three-dimensional mesh scaffold material is in the form of an injectable hydrogel.

[0124] In some embodiments, the three-dimensional mesh scaffold material is a nanostructure.

[0125] In the hydrogel, the concentration of the protein-responsive self-assembling peptide is ≥0.1 wt %, preferably 0.3-4 wt %, and more preferably 0.5-1 wt %.

[0126] The three-dimensional network scaffold material has more β-sheet structures than the self-assembling peptide.

[0127] In a fourth aspect, the present invention provides a composition comprising the protein-responsive self-assembling peptide of the first aspect and a proteinaceous substance or a mixed system comprising a proteinaceous substance.

[0128] The composition is in the form of a combination, or in the form of a combination, wherein the latter is to place the self-assembling peptide and the proteinaceous substance or a mixed system containing the proteinaceous substance in different containers respectively.

[0129] The protein is selected from proteins that can provide hydrogen ions under neutral physiological conditions; preferably, the protein is independently selected from proteins with an isoelectric point (PI) value lower than 7.0 (preferably 3.4-6.05).

[0130] In some embodiments, the protein is selected from laminin, fibronectin, fibrinogen, globulin, hemoglobin, vitronectin, transferrin, or a combination comprising one or more thereof, or a mixed system comprising one or more thereof, wherein the mixed system includes but is not limited to complete cell culture medium, serum-free culture medium, animal tissue, cell preservation medium or other cell or drug delivery system.

[0131] In some embodiments, the mixed system containing protein substances is serum, plasma, cell culture medium, animal and plant tissue fluid, etc.

[0132] The present invention unexpectedly discovered that when one or more protein substances are combined, or when a mixture of one or more of these substances is used, a more stable three-dimensional network scaffold structure is formed. 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.

[0133] The resulting hydrogel also exhibits self-repair capabilities. Mechanical forces can disrupt the hydrogel's gel state and restore it to its gel state after the mechanical force is removed. This recovery time is no more than 10 minutes, and the hydrogel's storage modulus after recovery is at least 70%, preferably at least 85%, and more preferably at least 95% of its pre-destruction value.

[0134] In a fifth aspect, the present invention provides the use of the protein-responsive 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 composition of the fourth aspect in one or more selected from the following: regenerative medicine and tissue regeneration; 2D and 3D cell culture and storage; drug delivery; wound healing; implantable materials; gene therapy; stem cell therapy; and medical cosmetology.

[0135] The hydrogel of the present invention is safe and convenient to prepare, and does not require adjustment of the pH value, temperature, light, salt or ion components of the system. Several endogenous proteins commonly found in the biomedical field can trigger self-assembly to form a gel, thereby better ensuring the biocompatibility of the hydrogel of the present invention.

[0136] The protein-responsive self-assembling peptides provided by the present invention, and the hydrogels prepared therefrom, can be used for in vitro three-dimensional culture and storage, establishing cell models, loading cells, organs, or organoids, and injecting them into animals or humans for tissue repair. They can also be used as wound dressings, hemostatic materials, etc., or as carriers for sustained-release drugs or functional factors, or as cell preservation materials in biotherapy, tissue engineering, and regenerative medicine. In summary, the protein-responsive self-assembling peptides of the present invention, and the hydrogels prepared therefrom, have a wide range of applications and are safe and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1A is a macroscopic photograph of a solution of a mixture of protein-responsive self-assembling peptides and proteins of the present invention, wherein tube 1 contains a mixture of protein-responsive self-assembling peptides and γ-globulin; tube 2 contains only a solution of protein-responsive self-assembling peptides, and the concentration of protein-responsive self-assembling peptides in both centrifuge tubes is 0.3 wt%; Figure 1B shows that using tissue fluid as an initiator, the self-assembling peptides can form a hydrogel, which remains in a gel state after being squeezed out of a syringe.

[0138] FIG2 is a TEM image of a mixture solution of the protein-responsive self-assembling peptide and protein of the present invention.

[0139] FIG3 is a circular dichroism spectrum of a mixture solution of the protein-responsive self-assembling peptide and protein of the present invention.

[0140] 4A-4B show the fluorescence changes of thioflavin T in the self-assembling peptide fiber hydrogel formed with fibrinogen, transferrin, and γ-globulin as initiators.

[0141] FIG5 shows the effect of different sequence structures of self-assembling peptides on the red blood cell support of the three-dimensional mesh scaffold.

[0142] Figure 6 shows the changes in rheological properties of different self-assembling peptide sequence structures, which are 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), and SEQ ID NO: 8 (I).

[0143] FIG7 shows the modulus results of a solution of a mixture of the protein-responsive self-assembling peptide of SEQ ID NO: 14 and protein after 30 minutes, wherein the concentration of the protein-responsive self-assembling peptide is 1 wt %.

[0144] FIG8 shows the modulus results of a solution of a mixture of the protein-responsive self-assembling peptide of SEQ ID NO: 18 and protein after 30 minutes, wherein the concentration of the protein-responsive self-assembling peptide is 1 wt %.

[0145] FIG9 shows a graph of shear thinning and recovery experimental results of the hydrogel of the present invention.

[0146] FIG10 shows a confocal laser scanning electron microscopy image of a hydrogel formed by the protein of the present invention and used to support cells, wherein the concentration of the protein-responsive self-assembling peptide is 0.1 wt %.

[0147] FIG11 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.

[0148] Figure 12 shows that 0.3wt.%, 0.5wt.%, and 0.7wt.% of the self-assembling peptide of the present invention were added to the cell culture system to induce self-assembly into a fiber network scaffold and culture liver cancer cells. The cell sphere diameters were counted on the third day, seventh day, eleventh day, and fifteenth day, and the characteristic cell spheres in the culture system were photographed.

[0149] Figure 13 shows that the addition of 0.3wt.% of the self-assembling peptide of the present invention to the cell culture system triggered its self-assembly into a fiber network scaffold and cultured porcine muscle satellite cells. The cell sphere diameters were counted on the first, second, third and fourth days, and the characteristic cell spheres on the first, second and fourth days in the culture system were photographed.

[0150] Figure 14 shows that the protein-responsive self-assembling peptide solution can stably disperse L-polylactic acid microspheres: A shows that the microspheres precipitate in the aqueous solution, but are evenly suspended and dispersed after mixing with the self-assembling peptide solution; B shows that the 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.

[0151] Figure 15 shows that the protein-responsive self-assembling peptide solution can stably disperse polycaprolactone: A shows that the microspheres precipitate in the aqueous solution, but are evenly suspended and dispersed after mixing with the self-assembling peptide solution; B shows that the 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 15B is mixed with tissue fluid, the protein-responsive self-assembling peptide / L-polylactic acid microsphere solution quickly forms a hydrogel. DETAILED DESCRIPTION

[0152] The present invention will be further described below with reference to specific embodiments. These embodiments are provided for illustration only and do not limit the scope of protection of the present invention.

[0153] The present invention provides protein-responsive self-assembling peptides and also provides a hydrogel prepared from the protein-responsive self-assembling peptides in the presence of a positively charged source protein substance or a mixed system containing a protein substance. The scaffold material in the form of a hydrogel is a hydrogel material having a three-dimensional network scaffold structure.

[0154] The protein capable of inducing self-assembly of protein-responsive self-assembling peptides is selected from laminin, fibronectin, fibrinogen, globulin, hemoglobin, vitronectin, transferrin, or a combination comprising one or more thereof, or a mixed system comprising one or more thereof, wherein the mixed system includes but is not limited to complete cell culture medium, serum-free culture medium, animal tissue, cell preservation fluid or other cell or drug delivery system.

[0155] Laminin (LN), primarily found in the basal lamina, is a non-collagenous glycoprotein unique to the basal lamina and one of the most important components of the extracellular matrix. As a widely distributed transmembrane glycoprotein on the cell surface, it forms the structural foundation for the adhesion of epidermal cells to the basal membrane. Laminin enhances intercellular adhesion and participates in specific adhesion processes between cells and between cells and the extracellular matrix. It also acts as a signaling molecule, transmitting various information to neighboring cells through interactions with the cell surface. It is crucial for maintaining the epidermis-dermis connection and the structure of the skin.

[0156] Fibronectin (FN) is a high-molecular-weight glycoprotein widely present in tissues and tissue fluid (extracellular matrix). It is composed of two subunits cross-linked by a disulfide bond at the C-terminus. It is synthesized by different cell types, such as fibroblasts, astrocytes, and early mesenchymal cells, and deposited in various forms on the cell surface, in the extracellular matrix, in the intercellular spaces, in the basement membrane, and in connective tissue. In the body's vital activities, it participates in cell migration, adhesion, proliferation, hemostasis, tissue repair, and embryonic development. It acts as a growth factor, promoting cell proliferation, inducing epidermal cells to form granulation tissue, and promoting the remodeling of the subepidermal basement membrane and normal keratinization. Fibronectin is also involved in numerous pathological processes.

[0157] Fibrinogen is a glycoprotein synthesized by hepatocytes that has coagulation function. Fibrinogen promotes platelet aggregation. Human fibrinogen is of great clinical significance. Decreased or deficient human fibrinogen can be seen in a variety of clinical conditions, including severe liver disease, disseminated intravascular coagulation, postpartum hemorrhage, major surgery, and traumatic bleeding.

[0158] Globulin is a serum protein found in the human body. Globulin is a common protein found in almost all plants and animals. In the human body, globulins can be divided into four types: α1, α2, β, and γ. In this application, γ-globulin is preferred.

[0159] Hemoglobin is a major protein in red blood cells. It is a quaternary protein composed of two α subunits and two β subunits. It is a special protein that transports oxygen within red blood cells and is the protein that makes blood red.

[0160] Vitronectin is a macromolecular protein located on the cell surface and in plasma. It is a major cell adhesion molecule that plays a structural and adhesive role in the cell fibrous matrix.

[0161] Transferrin receptor is a membrane transport protein involved in the transport of iron from plasma to cells. Transferrin receptor is composed of two identical 95 kDa independent subunits connected by two disulfide bonds.

[0162] Among them, fibrinogen, transferrin, gamma globulin, and hemoglobin are proteins that are widely present in the serum or plasma of organisms and are of great significance to wound coagulation, repair, and the normal immune function of organisms. The present invention has experimentally confirmed that the self-assembling peptides of the present invention can respond to these proteins and thus self-assemble to form hydrogels under the initiation of these proteins, which are then used as dressings for hemostasis and repair of wounds in vivo or in vitro. Laminin, fibronectin, and vitronectin are important components of the natural extracellular matrix that can promote cell adhesion and proliferation; for self-assembling peptides injected or implanted into the body, they can respond to the above proteins to form hydrogels in vivo. In addition, transferrin is also commonly used as an added component of serum-free culture medium in vitro, so that the self-assembling peptides can also self-assemble in an in vitro system to which this protein is added, further expanding the scope of application of the hydrogel of the present invention.

[0163] It is understandable that other protein substances for which detailed data are not provided in the examples of the present invention can also have the same or similar effects as long as they can provide positive charges, that is, achieve the self-assembly of the protein-responsive self-assembling peptides into the hydrogel of the present invention.

[0164] The protein-responsive self-assembling peptides of the present invention are in a neutral liquid state during use under human physiological conditions, thereby avoiding the risks caused by adjusting pH or introducing other exogenous substances, such as certain metal salt ions, specific proteins, etc.

[0165] The interaction between the acidic amino acids of the protein-responsive self-assembling peptides of the present invention and proteins triggers the self-assembly of the peptide aqueous solution into a hydrogel. Under neutral conditions, the addition of protein-like substances imparts a positive charge, neutralizing the negatively charged acid ions on the self-assembling peptide molecules, thereby reducing the repulsive forces between the self-assembling peptide molecules. The self-assembling peptide molecules then self-assemble through hydrophobic interactions and hydrogen bonds, ultimately forming a nanoscale three-dimensional network structure that constitutes the hydrogel.

[0166] 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 completely form a three-dimensional mesh scaffold material before adding a protein-like substance, and has a low viscosity, thereby maintaining the convenience of operating 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 a tight molecular stack cannot be formed. Even if the hydrophobic effect of the hydrophobic domain provides power for the aggregation of the self-assembling peptide, 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 in the solution. The β-turn structure reduces the interference of other side chains near the acidic amino acids. Therefore, the β-turn structure 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. Moreover, when the self-assembling peptide molecules approach each other during movement, they are given acceleration due to electrostatic interactions, which increases the kinetic energy of the entire system. Therefore, in a solution containing only self-assembling peptides under neutral conditions, the distribution of the self-assembling peptide molecules is relatively chaotic and cannot form a three-dimensional network scaffold material. Once protein-like substances are added to such a protein-responsive self-assembling peptide solution, when positive ions / groups are present, the carbonyl groups on the protein-responsive self-assembling peptides will form electrostatic interactions with them. The negative charges on the protein-responsive self-assembling peptide molecules are "shielded", the electrostatic repulsion between the molecules is reduced, and the hydrophobic interaction and hydrogen bonding cause the molecules to aggregate and distribute in an orderly manner, forming a three-dimensional network scaffold material.

[0167] Specifically, the hydrogen ions given by the proteinaceous substances in the present invention can attract each other with the acidic amino acids of the self-assembling peptide (negatively charged under near-physiological conditions and / or physiological conditions) due to electrostatic interaction, reducing and / or eliminating the electrostatic repulsion between the self-assembling peptide molecules before the addition of the protein, and providing a great driving force for the self-assembly of the self-assembling peptide under near-physiological conditions and / or physiological conditions. This drive is a non-covalent cross-linking and will not change the physical and chemical properties of these proteins themselves, so that they can still maintain and exert their original biological activity in the original system. This also shows that the hydrogel of the present invention can be obtained by initiation under the conditions of endogenous proteinaceous substances without introducing exogenous substances, and does not destroy the biological activity of the initiator in the original system. It is a safe and highly biocompatible hydrogel. The mass fraction ratio of proteinaceous substances to self-assembling peptides in the hydrogel of the present invention is (1-100): (100:1).

[0168] The concentration of the self-assembling peptide in the hydrogel of the present invention is preferably greater than 0.1 wt%, preferably within a concentration range of 0.3-4 wt%, and more preferably within a concentration range of 0.5-1 wt%. The self-assembling peptide of the present invention responds rapidly to proteinaceous substances. Within a short period of time, preferably within 30 minutes, preferably within 15 minutes, or even within 10 minutes after contact with the proteinaceous substance, the self-assembling peptide and the proteinaceous substance self-assemble into a hydrogel through non-covalent cross-linking, thus achieving gelation.

[0169] The protein-responsive self-assembling peptides of the present invention not only have the advantages of rapidly assembling to form hydrogels within a short period of time, but also exhibit the properties of shear thinning and rapid recovery to their original state after removal of external forces. This indicates that under the action of strong external mechanical forces, some non-covalent bonds within the hydrogel break, macroscopically manifesting as the gel state being disrupted and transformed into a solution. When the mechanical force disappears, these broken non-covalent bonds reconnect, macroscopically manifesting as the disrupted gel self-repairing to a state similar to its original gel state. Furthermore, the hydrogel recovery time does not exceed 10 minutes, and the storage modulus after recovery is at least 70% of the original value, and can even reach over 85% or even over 95% of the original value. Furthermore, the hydrogels of the present invention retain the ability to self-repair after undergoing multiple shear thinning events, meaning that shear thinning only temporarily disrupts the hydrogel's internal three-dimensional network structure, giving them the potential to form a gel in situ after injection. Shear thinning can be achieved using a variety of mechanical forces that exert shear or shear stress on the hydrogel, such as pipetting, centrifugation, shaking, injection, spraying, and filtration. Furthermore, the hydrogels of the present invention offer the advantage that the self-repair process after shear thinning can be terminated by dilution. Specifically, after shear thinning under mechanical force, dilution with a solvent prevents the hydrogel from returning to a gel state, rendering it liquid. This facilitates the separation of substances encapsulated within the hydrogel. For example, after culturing, storing, and transporting cells in a hydrogel, the cells can be separated from the hydrogel through shear thinning and sufficient dilution for harvesting.

[0170] The hydrogel of the present invention can avoid changing the existing use environment as much as possible when in use, which means that the hydrogel of the present invention can self-assemble in response to protein substances in the biological environment without introducing exogenous substances, and can be used for various purposes such as tissue repair, in vitro 3D cell culture, storage of cells and viruses, preparation of wound dressings, preparation of scaffolds required for tissue engineering, etc.; and it supports use in the form of injection; when used in vitro, it is beneficial to the replacement of cell culture medium and cell separation; when used in vivo, it can achieve in situ repair in the body and in vivo drug delivery, etc. For in vitro 3D cell culture, there is no need to add other components other than the complete cell culture medium; for some immune applications, protein substances within the application scenario can also be selected as components to trigger the self-assembly of self-assembling peptides to avoid introducing other proteins to interfere with the experiment. At the same time, the hydrogel of the present invention will not change the temperature and pH of the entire system during the self-assembly process and / or after the gel is formed, which brings great safety guarantees for its application in the biomedical field and can be widely used in the biomedical field.

[0171] In some embodiments, the hydrogel of the present invention is used for three-dimensional cell culture. After the self-assembling peptide solution of the present invention is directly mixed with the complete cell culture medium containing cells, it is transferred to culture equipment such as culture dishes, culture bottles, cell culture well plates, etc. to achieve three-dimensional cell culture. The external environmental conditions and culture medium required for three-dimensional culture are consistent with traditional two-dimensional cell culture, and no expensive equipment is required. Because the serum in the complete cell culture medium contains a variety of proteins, such as vitronectin, γ-globulin, etc., the self-assembling peptides of the present invention can respond and self-assemble into hydrogels, which can be used to support cells and promote cell proliferation. The hydrogel of the present invention is suitable for three-dimensional culture of various types of cells.

[0172] Glossary:

[0173] 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.

[0174] Hydrogels have the following properties:

[0175] 1. Good biological relevance: The polymer contains a large number of hydrophilic groups, which can absorb water dozens of times more than its own amount, and has the characteristics of swelling but not dissolving in water, and has good water retention capacity;

[0176] 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;

[0177] 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.

[0178] 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.

[0179] Peptide Self Assembly is a short chain of amino acids with a polar domain. When dissolved in a neutral solvent and physiological salt concentration, these self-assembling peptides spontaneously assemble into hierarchical nanostructures through hydrogen bonds, ionic bonds, hydrophobic interactions or van der Waals forces. Materials derived from these assemblies have the advantages of being non-toxic, non-immunogenic, non-thrombogenic, degradable and easily metabolized. At the same time, nanofibers have the same size scale as natural ECM fibers and can be easily designed to mimic the stiffness of various soft tissues. They can also be further functionalized by the attachment of cell-interacting peptide domains or cytokines and growth factors. Therefore, they can be used to design biologically relevant culture environments and improve the control of proliferating cell populations.

[0180] 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.

[0181] 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.

[0182] "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).

[0183] In the present invention, a "peptide" is an amino acid chain. In particular, a peptide is 2 to 40 amino acids in length.

[0184] In the present invention, "self-assembly" refers to the aggregation of self-assembling peptides into an ordered structure under normal environmental conditions.

[0185] 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.

[0186] 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.

[0187] The term "hydrophobicity" used in the present invention refers to a property of being inclined to repel water or being completely insoluble in water.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] "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.

[0192] 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.

[0193] Circular dichroism spectroscopy is the most widely used method for determining protein secondary structure and monitoring conformational changes of protein molecules induced by external conditions. Because the test is performed on liquids, the results obtained are closer to the secondary structure of proteins in real physiological environments. It is a fast, simple and relatively accurate method for studying protein conformation.

[0194] In the present invention, "response" and "trigger" are both used to indicate that the protein-responsive self-assembling peptides respond to different protein substances to form hydrogels, or that protein substances are used to trigger the protein-responsive self-assembling peptides to form hydrogels, and the two have the same meaning.

[0195] The present invention is further explained below through specific examples. Since the principle of the protein-responsive self-assembling peptides involved in the present invention to form hydrogels in response to different proteins is consistent, in multiple embodiments, the sequences of different protein-responsive self-assembling peptides are used as examples to provide verification data on the hydrogel properties and functions of the present invention. It is understandable that other self-assembling peptides that conform to the molecular structure of the self-assembling peptides of the present invention and for which detailed data are not provided in the present invention also have the same or similar effects. The amino acids mentioned in the present invention are characterized by single-letter abbreviations, which are generally accepted in the technical field.

[0196] Example 1. Synthesis of self-assembling peptides

[0197] The self-assembling peptide of the present invention was synthesized by a standard solid-phase peptide synthesis method. The amino acid sequence of the self-assembling peptide is shown in SEQ ID NO. 1-32 in the sequence listing. It was identified as the target self-assembling peptide by high performance liquid chromatography and mass spectrometry.

[0198] Example 2. Preparation of self-assembling peptides

[0199] Prepare a 2 wt% self-assembling peptide solution: add ultrapure water to an appropriate amount of the powder of the self-assembling peptide of SEQ ID NO. 1-32 obtained in Example 1, use a small amount of alkaline solution to assist in the complete dissolution of the self-assembling peptide, and after dissolution, use a trace of dilute hydrochloric acid and a weak alkaline solution to adjust the pH of the solution to neutral (for example, from about 6.5 to about 8, from about 7.0 to 7.5, and further adjust to about 7.0 to 7.2). The resulting solution is kept at 121 ° C for 30 minutes and then cooled to room temperature to obtain a mother solution of the self-assembling peptide shown in SEQ ID NO. 1-32, and stored at 4 ° C for use. When used, the self-assembling peptide mother solution is diluted with PBS buffer or ultrapure water to obtain a self-assembling peptide solution of SEQ ID NO. 1-32 of the desired concentration. For example, a 0.2 wt% self-assembling peptide solution is obtained by diluting an appropriate amount of 2 wt% self-assembling peptide mother solution 10 times with PBS buffer.

[0200] Example 3: Preparation of hydrogel

[0201] The self-assembling peptide solution of SEQ ID NO. 1-32 obtained in Example 2 is mixed with the protein solution, and the pH of the mixed solution is adjusted to neutral (e.g., about 6 to about 8, preferably about 6.5-7.5, preferably about 7-7.5) to obtain the hydrogel of the present invention. The protein is dissolved in a phosphate buffer (neutral pH) or a cell basal culture medium to obtain a protein solution. The mass fraction ratio of the self-assembling peptide to the protein in the mixed solution is (1-100): (100-1). According to this method, a hydrogel of any concentration of the self-assembling peptide of the present invention can be prepared.

[0202] Taking laminin (PI of approximately 4.94-6.05), fibronectin (PI of approximately 5.25), fibrinogen (PI of approximately 4.8), transferrin (PI of approximately 3.4-3.5), γ-globulin (PI of approximately 5.8), vitronectin (PI of approximately 5.47), collagen (PI of approximately 7.5-7.8), and hemoglobin (PI of approximately 7.23) as examples, 1 wt% solutions of laminin, fibronectin, fibrinogen, transferrin, γ-globulin, vitronectin, hemoglobin, and human collagen were prepared (the solvent was phosphate buffer, pH was neutral) and mixed with the 1 wt% self-assembling peptide mother solution obtained in Example 2 at a volume ratio of 1:1 to obtain the mixed systems of the Examples and Comparative Examples. The responses of each protein to the self-assembling peptides shown in each sequence are shown in Table 1.

[0203] The results in Table 1 show that the self-assembling peptides of the present invention can form hydrogels in response to proteins with an isoelectric point below 7.0 (such as laminin, fibronectin, fibrinogen, transferrin, gamma globulin, and vitronectin). In contrast, the self-assembling peptides of the present invention can only form weaker hydrogels (such as hemoglobin) or fail to form hydrogels (such as human collagen) when triggered by proteins with an isoelectric point above neutral.

[0204] The results in Table 1 also show that the self-assembling peptides of the present invention can also form hydrogels in response to compositions and mixtures containing proteins with an isoelectric point lower than 7.0, such as natural serum and plasma.

[0205] Using SEQ ID NO. 24 (FIIIVGSIIGPGGEGPGGE) as an example of a self-assembling peptide and γ-globulin as an example of a protein, the resulting hydrogel is shown in Figure 1A. The self-assembling peptide solution is in liquid form. After rotating the centrifuge tube 90°, the liquid surface flows parallel to the ground (tube 2 in Figure 1A), maintaining a solution state. In the presence of γ-globulin, the self-assembling peptide self-assembles into a hydrogel in response to the γ-globulin (tube 1 in Figure 1A). The centrifuge tube is rotated 90°. Due to the formation of the hydrogel, the fluidity decreases, but the liquid surface remains perpendicular to the ground. Figure 1B shows that using tissue fluid as an initiator, the self-assembling peptide can form a hydrogel, which remains in a gel state after extrusion through a syringe. The self-assembling peptides shown in SEQ ID NOs. 1-32 also exhibit similar or equivalent effects in forming hydrogels in response to other proteins (such as laminin, fibronectin, fibrinogen, transferrin, γ-globulin, vitronectin, or mixtures thereof) (indicated by √ or ~).

[0206] For example, a self-assembling peptide represented by the amino acid sequence LLLLLGSVLGPAGEGPAGE (SEQ ID NO: 29) rapidly formed a hydrogel upon mixing a 2% concentration of the self-assembling peptide with an equal volume of human tissue fluid. This hydrogel could be drawn up with a syringe and remained in a gel state after injection and extrusion. This also demonstrated similar or equivalent effects for peptide hydrogel materials formed in response to other types of triggering substances.

[0207] Example 4: Structural characterization of hydrogel materials

[0208] Experiment 1: Transmission Electron Microscopy (TEM)

[0209] Experimental method: Preparation of self-assembling peptide solution for TEM experiment: The 0.2 wt % self-assembling peptide solution obtained in Example 2 was diluted with ultrapure water to a self-assembling peptide concentration of 0.002 wt %.

[0210] Preparation of protein solution for TEM experiment: Laminin, fibronectin, fibrinogen, transferrin, γ-globulin, and vitronectin were added to ultrapure water to a protein concentration of 0.002 wt%.

[0211] Preparation of self-assembling peptide samples: The 0.2wt% self-assembling peptide solution obtained in Example 2 was diluted to 0.002wt% with ultrapure water and incubated at 37°C for one hour. 10μL was placed on a 300-mesh carbon support film copper grid (purchased from Beijing Xinxing Bairui Technology Co., Ltd.) and allowed to stand at room temperature until completely dry. After complete drying, 10μL of 2wt% phosphotungstic acid negative staining solution was dropped onto the copper grid supporting the sample. After staining for 60 seconds, the excess stain was removed and the staining step was repeated three times. After staining, the copper grid was allowed to stand at room temperature to dry.

[0212] Preparation of the hydrogel sample of the present invention: 0.002wt% self-assembling peptide solution and 0.002wt% protein solution were mixed in a volume ratio of 1:1, incubated at 37°C for one hour, and 10μL of each was placed on a 300-mesh carbon support film copper mesh (purchased from Beijing Xinxing Bairui Technology Co., Ltd.), and allowed to stand at room temperature. During the standing process, the water in the mixed system evaporated, allowing the self-assembling peptide and protein to gradually concentrate to form a hydrogel. After standing until completely dry, 10μL of 2wt% phosphotungstic acid negative staining solution was dropped on the copper mesh carrying the sample, and the excess stain was absorbed after 60 seconds of staining. This staining step was repeated three times. After staining is completed, the copper mesh was allowed to stand at room temperature to dry.

[0213] All samples were observed and photographed using a Talos G2 200X transmission electron microscope. Taking the hydrogels formed by the self-assembling peptide represented by SEQ ID NO. 13 (IIIIIGOGIIGPGGEGPGGE) with fibrinogen, transferrin, and γ-globulin as an example, the imaging results are shown in Figure 2.

[0214] When the self-assembling peptide molecules exist alone (see the control group), they can also be assembled into fibers, but the fibers are of different lengths and unevenly distributed, with an overall short length, and the fibers are almost not interwoven into bundles. In comparison, it can be seen that: under the same concentration of self-assembling peptides, due to the response to proteins (see the fibrinogen triggering group, the transferrin triggering group, and the gamma globulin triggering group), the number and length of fibers formed by the self-assembling peptide molecules are significantly improved, the degree of curling decreases, and the arrangement of the fibers also changes. The fiber distribution is more uniform, and cross-linking occurs between the fibers to aggregate into bundles. The fiber bundles are entangled with each other and interwoven into a tight and uniformly spaced three-dimensional network structure. The above phenomenon shows that under the initiation of protein, the self-assembling peptides will undergo a more stable and orderly self-assembly, thereby forming a more dense, stable, strong and uniformly porous nanofiber three-dimensional network structure, which can efficiently retain water and form a well-supported structure. This structure is conducive to supporting cells during cell culture and building a three-dimensional growth space for cells.

[0215] 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.

[0216] Experiment 2: Circular Dichroism (CD) Analysis

[0217] Experimental method: Preparation of self-assembling peptide solution for CD detection: The 0.2 wt % self-assembling peptide solution obtained in Example 2 was diluted with PBS buffer to a peptide concentration of 0.02 wt %.

[0218] 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%.

[0219] Preparation of self-assembling peptide samples: The 0.2 wt % self-assembling peptide solution obtained in Example 2 was diluted with PBS buffer to a peptide concentration of 0.01 wt %, and incubated at 37° C. for one hour.

[0220] To prepare the 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 and incubated at 37°C for one hour to obtain a mixture with 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.

[0221] 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 hydrogels formed by the self-assembling peptide represented by SEQ ID NO. 13 (IIIIIGOGIIGPGGEGPGGE) with fibrinogen, transferrin, and gamma globulin, respectively, are shown in Figure 3.

[0222] The results of circular dichroism detection show that the responsiveness of self-assembling peptides to proteins is manifested as changes in secondary structure. Figure 3 shows that when the self-assembling peptides exist alone (see control group) under a neutral environment, the secondary structure of the nanofibers assembled therein is mainly β-folding. In the system where proteins are introduced respectively, it can be seen that the positive peak near 200nm in the circular dichroism spectrum shifts to the right to varying degrees. This shows that compared to when the self-assembling peptides exist alone and are not triggered, the response to proteins increases the β-turn in the secondary structure of the nanofibers assembled by the self-assembling peptides, providing a favorable basis for forming more β-folding 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, gamma globulin triggering group). This is the embodiment of the characteristic polymeric structure formed by the participation of proline or hydroxyproline in the assembly of the self-assembling peptides on the fiber secondary structure. This shows that the response to these three substances makes the secondary structure of the fibers assembled by the self-assembling peptides 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 can assemble into nanofibers with specific secondary structures by themselves, their responsiveness to proteins enables the peptides or their derivatives 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 seen in Experiment 1.

[0223] Experiment 3: Thioflavin T fluorescence experiment

[0224] 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 to 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%) solutions of the self-assembling peptides were mixed with 0.072 wt% of a protein-based initiator at a 1:1 volume ratio. Ultrapure water was used instead of the protein-based initiator as a control. After standing for 15 minutes, the solution was 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.

[0225] Experimental Results: CD spectral data were verified 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 gel, and their content was positively correlated with the concentration of the self-assembling peptide, indicating that the self-assembling peptide is a concentration-dependent self-assembling peptide (Figure 4A). This is because the increase in peptide monomers facilitates intermolecular contact, thereby increasing the degree of assembly. The β-sheet content of the gel-generated group after adding protein-based initiators was much higher than that of the gel group (Figure 4B). CD and ThT staining data indicate that under neutral pH conditions, the supramolecular polymers generated by protein-activated self-assembling peptides exhibit a typical β-sheet structure.

[0226] 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

[0227] Experiment 1: Verification of red blood cell support by different sequences under liquid conditions

[0228] 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.

[0229] 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.

[0230] Using a 2ml transparent glass bottle, 2mL of self-assembling peptide solution (containing fibronectin with a final concentration of 0.1wt%) and a blank group (containing no self-assembling peptide solution and 0.1wt% fibronectin) were prepared with the four materials, respectively. 5×10 9 The final concentration of red blood cell stock solution was 1×10 8 , pipetting evenly. Photos were taken every four hours to compare the red blood cell support effects of different materials. Because the hydrogel formed by the self-assembling peptides of the present invention is a water-swelling system, the low-concentration peptide network scaffold, even in solution, still demonstrates the peptide hydrogel's support capacity.

[0231] Experimental conclusion: Among the self-assembling peptides with similar amino acid sequences, hydroxyproline and alanine have a significant impact on the cell-supporting ability of hydrogels or the scaffold solutions formed by the swelling of hydrogels. As shown in Figure 5, at high concentrations (0.1 wt%, 0.05 wt%), all four self-assembling peptide solutions can effectively support red blood cells at a concentration of 1×10 8 ; However, at low concentrations (0.01 wt%), hydroxyproline, alanine, and glutamate in the hydrophilic domain also showed a significant impact on the red blood cell-supporting ability. The four self-assembling peptide solutions showed obvious sedimentation at 8 hours, 24 hours, 32 hours, and 48 hours respectively. The supporting ability of the four self-assembling peptides from weak to strong is: 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 response to fibronectin, compared with SEQ ID NO:1, glutamate in the hydrophilic region of SEQ ID NO:2, glutamate and hydroxyproline in the hydrophilic region of SEQ ID NO:3, and glutamate, hydroxyproline, and alanine in the hydrophilic region of SEQ ID NO:7 successively showed an increasingly stronger enhancing effect on the cell-supporting ability. Hydroxyproline is abundant in the collagen tissues 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-assembling peptides can be significantly enhanced through the enhanced hydrogen bond network between self-assembling peptides, thereby improving the stability of the self-assembled scaffold and enhancing the cell-supporting function. At the same time, by replacing a few glycines with alanines and aspartic acids with glutamic acids in the β-sheet structure in the hydrophilic region, the hydrophobicity of the amino acid side chains can be enhanced without destroying the β-turn structure in the hydrophilic region, and the stability of the self-assembled material can also be increased by improving the hydrophobic interaction between polypeptides, thereby making the scaffold material more supportive to cells.

[0232] Experiment 2: Rheological experiment, influence of the secondary structure and amino acid sequence of self-assembling peptides [[ID=,7]]

[0233] 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 (IIIIIIGSIIGOGAEGPGGVGPG GV).

[0234] 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.

[0235] 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.

[0236] 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 Figure 6) that SEQ ID NO:7 (IIIIIGSIIGOGAEGPGGV)>SEQ ID NO:3 (IIIIIGSIIGOGGEGPGV)>SEQ ID NO:2 (IIIIIGSIIGPGGEGPGGV)≥SEQ ID NO:1 (IIIIIIGSIIGPGGDGPGGV); this experimental phenomenon is consistent with the results of Experiment 3, that is, the replacement of glycine (G) with alanine (A) in hydroxyproline and hydrophilic regions can enhance the mechanical strength of the hydrogel formed by the present invention, thereby enhancing the support function of the scaffold material. At the same time, 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 6, 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.

[0237] 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:3 (IIIIIGSIIGOGGEGPGGV), 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.

[0238] Example 6: Determination of hydrogel mechanical properties

[0239] Experiment 1: Determination of mechanical strength of hydrogel

[0240] Experimental method: Preparation of self-assembling peptide solution for dynamic rheological test: Take the self-assembling peptide mother solution prepared in Example 2 with a concentration of 2 wt %.

[0241] Preparation of protein solutions for dynamic rheological testing: Use ultrapure water to prepare laminin solution, fibronectin solution, fibrinogen solution, vitronectin solution, γ-globulin solution, transferrin solution, hemoglobin solution, and human collagen solution with a protein concentration of 2 wt%.

[0242] Preparation of self-assembling peptide samples: The 2 wt % self-assembling peptide stock solution of Example 2 was diluted to 1 wt % with ultrapure water.

[0243] Preparation of hydrogel samples: 2 wt% of the self-assembling peptide mother solution was mixed with the above 2 wt% of laminin, fibronectin, fibrinogen, vitronectin, γ-globulin, transferrin, hemoglobin, and human collagen solutions in a 1:1 volume ratio to obtain a mixed system with a final concentration of 1 wt% of the self-assembling peptide.

[0244] To simultaneously measure the gel formation rate and strength of the mixed system, 200 μL of each sample was immediately added to the 20 mm diameter faceplate of a MARS 60 rheometer upon preparation. The final modulus was recorded after a 30-minute time scan. The distance between the rotor and the faceplate was 500 μm, the shear strain was 1%, and the frequency was 1 Hz. The entire test was completed at 37°C. The results of the mixed system formed by the self-assembling peptide represented by SEQ ID NO. 14 (FIFIFGTVIGPGGEGOGGV) with laminin, fibronectin, fibrinogen, vitronectin, transferrin, gamma globulin, hemoglobin, and human collagen are shown in Figure 7.

[0245] The results show that in each sample with a self-assembling peptide concentration of 1wt%, when the self-assembling peptide exists alone, although its storage modulus is higher than the loss modulus, there is no obvious difference, and both are lower than 2Pa (not shown in the figure), and the macroscopic appearance is liquid rather than solid; but when protein is present in the sample, the storage modulus (G') of the mixed system self-assembling peptide material of the present invention rises rapidly within 5 minutes, which is significantly higher than the loss modulus (G"), and forms a high-strength hydrogel with an elastic modulus greater than 100Pa within 30 minutes (Figure 7 shows G' and G" at 30 minutes). This shows that the self-assembling peptide molecules of the present invention are rapidly assembled to form a hydrogel with a certain mechanical strength under the initiation of positively charged natural substances (especially proteins with an isoelectric point PI of less than 7.0). However, due to its high isoelectric point, hemoglobin can only form a weaker gel, and the mixed solution of collagen cannot form a gel at all.

[0246] Experiment 2: Determination of mechanical strength of the hydrogel of the present invention

[0247] Experimental method: Same as Experiment 3, taking the mixed system formed by the self-assembling peptide represented by SEQ ID NO. 18 (IVIVIGSIIGOGGDGPGGV) and laminin, fibronectin, fibrinogen, and human serum albumin as an example, the results are shown in Figure 8.

[0248] The storage modulus (G') and loss modulus (G") measured after the polypeptide solution was mixed with different initiating proteins for 30 minutes are shown in Figure 8. The mechanical properties of the hydrogels formed by different proteins showed obvious differences. In all groups with the addition of the initiating protein of the present invention, hydrogels with a storage modulus greater than 300 Pa were observed to be formed in the mixed system, which is at least twice the storage modulus of the hydrogel formed by human serum albumin. This shows that the responsive hydrogels formed by the self-assembling peptides of the present invention and laminin, fibronectin, and fibrinogen have stronger mechanical strength, that is, stronger supporting function, than the responsive hydrogels formed by the self-assembling peptides and human serum albumin.

[0249] Experiment 3: Determination of the reassembly characteristics of the hydrogel after deformation

[0250] Experimental method: A shear thinning test was performed to verify the reassembly performance of the hydrogel of the present invention after deformation.

[0251] Preparation of hydrogel samples: 1 wt% self-assembling peptide solution was mixed with 1 wt% laminin, fibronectin, fibrinogen, vitronectin, γ-globulin, transferrin, hemoglobin, and human collagen solutions in a 1:1 volume ratio to obtain a mixed system with a final concentration of 1 wt% self-assembling peptide.

[0252] During the test, 200 μL of the mixed system was added to the 20 mm diameter faceplate of a MARS 60 rheometer and a time sweep was performed. Shear thinning was then performed for 20 seconds with a rotor-faceplate spacing of 500 μm, a shear strain of 100%, and a frequency of 1 Hz. Shear thinning was then stopped, the shear strain immediately adjusted to 1%, and a time sweep test was immediately performed to monitor the recovery of the hydrogel's mechanical strength. Each 20-second shear thinning test constituted a single set of experiments, which were repeated twice to examine the reassembly ability of the hydrogels of the present invention after multiple external force-induced deformation. Using a mixed system formed by the self-assembling peptide represented by SEQ ID NO. 13 (IIIIIGOGIIGPGGEGPGGE) and tissue fluid (containing a large amount of proteins such as laminin, fibronectin, and vitronectin) as an example, the results are shown in Figure 9.

[0253] The results in Figure 9 show that the hydrogel self-assembled in response to tissue fluid exhibits the characteristic of being able to quickly re-self-assemble after being damaged and deformed by external force, and this characteristic does not disappear as the number of damages increases (Figure 9). When subjected to 100% shear strain, some non-covalent bonds inside the hydrogel, such as hydrogen bonds, break, causing its mechanical strength to decrease and become liquid; when the external force disappears, the partially destroyed nanofiber clusters will quickly approach and reassemble, quickly restoring their original mechanical strength. Figure 9 shows that after experiencing multiple shear thinning, the hydrogel of the present invention can still re-self-assemble, and the self-assembly time and the strength of the reorganized hydrogel are extended with only a small loss as the number of shear thinning increases.

[0254] Because laminin, fibronectin, vitronectin, and other proteins are abundant in tissue fluid, the protein-responsive peptides of the present invention can be rapidly activated to form a hydrogel. The resulting hydrogel exhibits shear-thinning and self-repairing properties and is injectable. When used in cell culture, the hydrogel of the present invention enables multiple pipetting transfers, facilitating cell packaging and fluid replacement. Furthermore, when used in the biomedical field, it allows for multiple injections.

[0255] 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.

[0256] Example 7: Application of the peptide hydrogel material of the present invention in the field of biomedicine

[0257] Experiment 1: The supporting effect of the hydrogel of the present invention on cells

[0258] Experimental method: Adherent HepG2 liver cancer cells were cultured in a cell culture flask in a mild and humid environment at 37°C and 5% CO2 using high-glucose DMEM medium containing 10% serum. After culture, the cells were collected and resuspended in PBS buffer solution to obtain a concentration of 5×10 5 cells / mL of cell suspension. The prepared cell suspension and Calcein-AM / PI staining solution were mixed evenly in a volume ratio of 2: 1 and incubated in the dark at 37°C for 15 minutes. A hydrogel containing fibrinogen, transferrin, and γ-globulin prepared in advance was added to a glass-bottomed culture dish (see Example 3 for the preparation method). The cell suspension after incubation in the dark was then added and mixed to make the total volume in the glass-bottomed culture dish 200 μL and the final concentration of the self-assembling peptide 0.1 wt%. When preparing the control group sample, an equal volume of PBS buffer was used to replace the hydrogel material of the present invention in the experimental group. Subsequently, each experimental sample was observed using an LSM 980 with Airyscan2 fast super-resolution laser confocal microscope. Taking the mixed system formed by the self-assembling peptide shown in SEQ ID NO.13 (IIIIIGOGIIGPGGEGPGGE) and fibrinogen, transferrin, and γ-globulin as an example, the results are shown in Figure 10.

[0259] The results in Figure 10 show that in the control group without the hydrogel of the present invention, 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 peptides were introduced into the system to form a hydrogel that self-assembled in response to fibrinogen, transferrin, and γ-globulin, the cells in the system were supported by the three-dimensional network scaffold formed by the self-assembling peptides, showing a three-dimensional distribution in a 3D environment.

[0260] Experimental Conclusion: This experimental phenomenon demonstrates that the hydrogel material of the present invention, namely, the three-dimensional network scaffold formed by the self-assembling peptides in response to proteins, can effectively support cells, facilitate the growth and cultivation of cells in three dimensions in vitro, and prevent them from sinking and adhering to the wall, thereby allowing them to grow in a two-dimensional environment. Furthermore, the entire initiation process is mild, requires no additional substances, and is effective, convenient, and harmless to cells. This demonstrates the enormous potential of the polypeptide material of the present invention in the fields of cell culture and biomedicine.

[0261] 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.

[0262] Experiment 2: Application of 3D Cell Storage

[0263] Experimental method: This experiment takes the storage of mouse mesenchymal stem cells at 4°C as an example. The isolated mouse mesenchymal stem cells were pipetted evenly and 10 ml of equal volume was dispensed into cryopreservation tubes and pipetted evenly to make the cell concentration between 1×10 6 In the 3D group, mouse serum and 1% double-antibody storage solution in 50% MAP + 50% SFEM were added to the peptide SEQ ID NO: 20 (FLIVIGOGIIGOGGEGPGGE) to a final concentration of 0.05%. In the 2D group, mouse serum and 1% double-antibody storage solution in 50% MAP + 50% SFEM were added, and then the cells were stored in a 4°C refrigerator.

[0264] Cell viability test:

[0265] (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;

[0266] (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.

[0267] Experimental results: In order to understand the changes in the survival rate of mesenchymal cells during the storage period, we used the Calcein-AM / PI live / dead double staining kit to double-stain them and detected them using a flow cytometer. As shown in Figure 11, 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.

[0268] 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.

[0269] Experiment 3: Application of 3D Culture of Liver Cancer Cells

[0270] 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 adherent cells HepG2 were cultured in the self-assembling peptide culture system of the present invention induced by high sugar culture medium. Whether cell spheres can be formed was used to verify that cell culture similar to that in organisms can be achieved in vitro.

[0271] Prepare the concentration of 1×10 6 The concentration of 1×10 cells / mL cell suspension was prepared by 5 Taking SEQ ID NO:3 (IIIIIGSIIGOGGEGPGGV) as an example, the concentration obtained is 1×10 5 A cell suspension of 10 cells / mL was inoculated into a 96-well plate containing the self-assembling peptide of the present invention (the final concentration of the self-assembling peptide in each well was 0.3 wt.%, 0.5 wt.%, and 0.7 wt.%). The culture medium in the upper portion of the plate was removed and replaced with fresh medium every 3 days during the culture period.

[0272] After 3, 7, 11, and 15 days of culture, the diameters of 50 cell spheres in the culture system were observed and recorded under an inverted microscope. The cell sphere diameter data collected on these three days were averaged and plotted into a graph (Figure 12). The HepG2 cells cultured in the self-assembling peptides of the present invention were tested to see whether they could achieve long-term in vitro proliferation, and characteristic cell spheres were photographed (Figure 12). As shown in Figure 12, in a culture system containing 0.3wt.%, 0.5wt.%, and 0.7wt.% of the triggered self-assembling peptides of the present invention, HepG2 cells were supported and subsequently grew and proliferated in the form of cell spheres. As the culture time prolonged, the cell spheres became increasingly compact and their diameters increased at a similar rate, indicating that HepG2 cells were able to proliferate stably at a uniform rate over a 15-day culture period. That is, the culture system of the present invention, which is triggered by a mixed system containing protein-containing substances and contains self-assembling peptides, can be used for long-term in vitro three-dimensional cell culture. The relatively uniform cell proliferation rate also indicates that it is in good condition during the culture period and does not cause damage to the cells.

[0273] 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.

[0274] Experiment 4: Application of 3D Culture of Porcine Muscle Satellites

[0275] The culture of porcine muscle satellite cells was similar to the 3D culture of liver cancer cells in Experiment 3. The concentration was prepared as in Experiment 2, 1×10 6 The concentration of 1×10 cells / mL cell suspension was prepared by 5 Taking SEQ ID NO: 21 (IVIVIGOGIIGOGGDGOGGV) as an example, the concentration obtained is 1×10 5 Cell suspensions of 10 cells / mL were inoculated into 96-well plates containing the self-assembling peptides of the present invention (the final concentration of the self-assembling peptides in each well was 0.3 wt.%). The culture medium in the upper portion of the plate was removed and replaced with new medium every 3 days during the culture period.

[0276] The diameters of 50 cell spheres in the culture system were observed and recorded under an inverted microscope after 1, 2, 3, and 4 days of culture. The averaged sphere diameter data collected over these three days were calculated and plotted (Figure 13). This was used to test whether porcine muscle satellite cells cultured in the self-assembling peptides of the present invention could achieve long-term in vitro proliferation, and characteristic spheres were photographed (Figure 13). As shown in Figure 13, in a mixed culture system containing 0.3 wt.% of the polypeptides of the present invention and a protein-containing substance, porcine muscle satellite 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 consistent rate. This indicates that porcine muscle satellite cells were able to proliferate stably and uniformly over the four-day culture period. Specifically, the self-assembling peptides of the present invention, after being triggered by the protein-containing mixture, formed a 3D support structure, which 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 were not damaged.

[0277] Experiments 1-4 of this example demonstrate that the culture system of the protein-based self-assembling peptides of the present invention has excellent cell-supporting capacity. Experiment 2 also demonstrates that the protein-based self-assembling peptides of the present invention have good biocompatibility and can be used for cell storage. Experiments 3 and 4 respectively demonstrate that the addition of the self-assembling peptides of the present invention to cell culture media containing natural protein-based substances can highly replicate the in vivo cell growth environment, enabling long-term three-dimensional cell culture in vitro.

[0278] Example 8: Application of the hydrogel of the present invention as a dispersed filler for medical aesthetics

[0279] 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 stored at room temperature for one month. The obtained injections were uniformly suspended, with no obvious solid-liquid separation. After adding 1 / 5 tissue fluid, hydrogel formation was observed (Figure 14C).

[0280] 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, there 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).

[0281] In addition, except for SEQ ID NOs. 25 and 27, the self-assembling peptides represented by SEQ ID NOs. 1-7 and 9-32 of the present invention can have the same function on a variety of other medical aesthetic regeneration microspheres (Table 2).

[0282] Table 2 Common medical aesthetic microspheres:

[0283] 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.

[0284] 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.

[0285] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention are intended to be included in the scope of protection of the present invention.

Claims

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

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

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

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

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

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

10. The protein-responsive self-assembling peptide according to claim 1, wherein the C-terminus of the hydrophilic domain is modified with a reagent or group selected from the 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 modification.

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

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

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

14. The protein-responsive self-assembling peptide according to claim 1, further comprising a connecting domain, Preferably, the linker domain comprises 2-8 amino acid residues, preferably 4-5 amino acid residues, Preferably, the linker domain comprises amino acids with small side chains, amino acids with hydroxyl groups on their side chains and / or hydrophobic amino acids away from the hydrophobic region. Preferably, the small side chain amino acid is selected from glycine (G), alanine (A) and serine (S), the amino acid with a hydroxyl group on the side chain is selected from serine (S), threonine (T) and hydroxyproline (O), the hydrophobic amino acid away from the hydrophobic domain is selected from I, V, L, F and A, and the hydrophobic amino acids I, V, F, L, and A are interchangeable. Preferably, the linking domain has an amino acid sequence selected from the group consisting of: GSII (SEQ ID NO.: 89), GPOGI (SEQ ID NO.: 90, GPOGV (SEQ ID NO.: 91), GSGII (SEQ ID NO.: 92), GSVI (SEQ ID NO.: 93), GOII (SEQ ID NO.: 94), GPOGL (SEQ ID NO.: 95), OGII (SEQ ID NO.: 96) or GTVI (SEQ ID NO.: 97), wherein, S, T, and O can be interchanged with each other; More preferably, the connecting domain has an amino acid sequence selected from the group consisting of: GSII (SEQ ID NO.:89), GTII (SEQ ID NO.:98), GTVI (SEQ ID NO.:97), GOVI (SEQ ID NO.:99), GSVI (SEQ ID NO.:93), GSVL (SEQ ID NO.:100), GSGII (SEQ ID NO.:92), GSGVI (SEQ ID NO.:101), GOII (SEQ ID NO.:101) NO.:94), OGII (SEQ ID NO.:96), GOGVI (SEQ ID NO.:102) or GOGII (SEQ ID NO.:103). 15 . The protein-responsive self-assembling peptide according to claim 1 , having 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 scaffold material from the protein-responsive self-assembling peptide according to any one of claims 1 to 15, the method comprising the steps of inducing the self-assembling peptide to form the scaffold material using a proteinaceous substance or a mixed system containing a proteinaceous substance, Preferably, the method comprises the step of mixing the self-assembling peptide into a proteinaceous substance or into a mixed system comprising a proteinaceous substance; or the method comprises the step of injecting or implanting the self-assembling peptide into a mixed system comprising the proteinaceous substance.

17. The method according to claim 16, wherein the proteinaceous substance is selected from proteins that tend to donate hydrogen ions under neutral physiological conditions; Preferably, the proteins are independently selected from proteins having an isoelectric point (PI) value lower than 7.0 (preferably 3.4-6.05), More preferably, the proteins are independently selected from proteins having an isoelectric point (PI) value between 3.4 and 6.

05.

18. The method according to claim 16, wherein the proteinaceous substance is selected from laminin, fibronectin, fibrinogen, globulin, hemoglobin, vitronectin, transferrin, or a combination thereof; The mixed system containing protein substances is selected from serum, plasma, cell culture medium, animal and plant tissue fluid or animal tissue.

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

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 protein-responsive self-assembling peptide according to any one of claims 1 to 15 and the proteinaceous substance according to claim 16 or a mixed system comprising the proteinaceous substance.

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

Images