Protein-responsive self-assembling peptides and their applications

Abstract

The present invention provides a protein-responsive hydrogel and a method for preparing the same. The hydrogel comprises a nanofiber three-dimensional network structure formed by protein-induced self-assembly of a polypeptide or its derivative solution, and can be used for three-dimensional culture in vitro. It is useful for tissue repair, wound dressings, hemostatic materials, dispersion and in-tissue molding materials containing regenerative microspheres such as L-polylactic acid and polycaprolactone for cosmetic and medical use, carriers for the sustained release of drugs or functional factors, and cell preservation materials.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedical materials, and more particularly to protein-responsive peptide-based hydrogel biomaterials. [Background technology]

[0002] Self-assembling peptides have attracted attention due to their excellent biocompatibility and diverse functions. Research has shown that self-assembling peptides can spontaneously form structured aggregates in response to specific conditions. The self-assembly process of self-assembling peptides is primarily driven by a series of non-covalent interactions between self-assembling peptide molecules and their derivatives, including hydrogen bonding, electrostatic interactions, hydrophobic interactions, and π-π deposition. Self-assembling peptides can self-assemble into scaffolds with three-dimensional network structures, completing the solution-to-gel transition of the system and highly reproducing the biological structure of the natural extracellular matrix (ECM). They can also provide synergistic biochemical and biophysical cues to guide cell proliferation, migration, and differentiation, and are injectable. This is becoming increasingly important in the fields of cell culture, tissue engineering, and biomedicine. However, the self-assembly of self-assembling peptides is generally prepared under strict conditions and can only be initiated under certain environmental conditions, exogenous substances, or time limitations, such as specific temperatures (CN106083634A, the initiation temperature must be >40°C), pH (CN109776651A, the initiation must be under alkaline conditions), light irradiation (CN103992486A, the initiation must be by irradiation with a UV lamp with a wavelength of 300-400 nm), or other additional conditions (CN114507270A, the initiation must be via ultrasonic means). Self-assembly can only be achieved under certain conditions, such as the introduction of an enzyme (CN110325204A, which requires the addition of an enzyme as an initiator) or other exogenous substances (CN114344481A, which requires the addition of dimethyl sulfoxide, which poses a safety risk to living organisms, to promote dissolution during production), or a sufficiently long period of time (CN113416264A, which requires the production process to last 48 hours). These conditions require certain equipment, are detrimental to the survival or safety of cells or animals, and are unfavorable to the convenience of operation, limiting the scope of hydrogel use.On the other hand, hydrogels in which self-assembly is triggered by endogenous substances (e.g., substances present in the application scenario of the hydrogel) often have limited application scenarios due to the single starting material.

[0003] Therefore, the development of self-assembling peptides that can self-assemble to form nano-network structures under physiological conditions, cell preservation culture environments, and conditions where substances are widely present in the human body, particularly in the presence of in vivo proteins, and that can exert supporting and repair functions in solution, has very important future applications. Summary of the Invention

[0004] The present invention provides a protein-responsive self-assembling peptide solution system that can self-assemble to form a nano-network structure in response to the presence of proteins, particularly under a wide range of endogenous protein conditions, and that can exert supporting and repair functions in a solution state.

[0005] In a first aspect, the present invention provides a protein-responsive self-assembling peptide, the self-assembling peptide comprising a hydrophobic domain and a hydrophilic domain, the hydrophilic domain comprising at least two consecutive β-turn regions capable of forming a β-turn.

[0006] 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 one acidic amino acid.

[0007] In some embodiments, at least one β-turn region comprises a terminal acidic amino acid.

[0008] 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: X1X2X3, X1X2X3X4, X1X2X3X4X5, or X1X2X3X4X5X6, Here, X1, X2, X3, X4, X5, and X6 are amino acid residues, and in each β-turn motif, X1, X2, X3, X4, X5, and X6 are the same as or different from each other.

[0009] In some embodiments, the β-turn motif comprises one hydroxyproline (O), preferably, X2 is hydroxyproline (O).

[0010] In some embodiments, the hydrophilic domain comprises 2-8 β-turn regions.

[0011] In some embodiments, the hydrophilic domain comprises 2, 3, 4, 5, 6, 7, or 8 β-turn regions.

[0012] Preferably, the hydrophilic domain comprises 2-6 β-turn regions, more preferably 2-4 β-turn regions.

[0013] Preferably, the hydrophilic domain comprises two or three β-turn regions.

[0014] In some embodiments, the β-turn motif in the at least one β-turn region comprises or links one acidic amino acid.

[0015] In some embodiments, the β-turn motif in the at least one β-turn region comprises or links one glutamic acid (E), valine (V), leucine (L), isoleucine (I), aspartic acid (D), or lysine (K).

[0016] In some embodiments, the hydrophilic domain comprises two β-turn regions, the ends of which comprise the acidic amino acid E.

[0017] In some embodiments, the hydrophilic domain comprises two β-turn regions, one β-turn region having an acidic amino acid E at its end and the other β-turn region having an amino acid V or K at its end.

[0018] In some embodiments, the hydrophilic domain comprises two β-turn regions, the ends of which comprise the acidic amino acid D.

[0019] In some embodiments, the hydrophilic domain comprises two β-turn regions, one β-turn region having an acidic amino acid D at its end and the other β-turn region having an acidic amino acid V at its end.

[0020] In some embodiments, the hydrophilic domain comprises a β-turn motif where at least one X2 is hydroxyproline O, or the hydrophilic domain comprises a β-turn motif where at least one X2 is proline P.

[0021] In some embodiments, the hydrophilic domain comprises at least one X2=O β-turn motif.

[0022] In some embodiments, the hydrophilic domain comprises at least one β-turn motif where X1 is G.

[0023] In some embodiments, the hydrophilic domain comprises a β-turn motif where one X2 is O and one X2 is P.

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

[0025] In some embodiments, the hydrophilic domain comprises a β-turn motif in which two X2 are P.

[0026] In some embodiments, one or more of X1, X3, and X4 is glycine (G), and / or one or two of X3 and X4 is alanine (A).

[0027] In some embodiments, the β-turn motif comprises an amino acid sequence selected from the following: 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.:41), GOAA (SEQ ID NO.:42), GOG, or GOGV (SEQ ID NO.:43).

[0028] Preferably, the β-turn motif has an amino acid sequence selected from the following: 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).

[0029] In some embodiments, X1 and X4 form a hydrogen bond.

[0030] 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.

[0031] In some embodiments, the β-turn motif has an amino acid sequence selected from the following: 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).

[0032] The hydrophilic domain contains at least one β-turn motif containing an alanine, thereby improving the mechanical properties of the self-assembling peptide.

[0033] In some embodiments, the C-terminus of the hydrophilic domain may be modified with a reagent or group selected from carboxylic acid, thiol, ketoacid salt, nitrite, phosphonate, sulfite phosphate, carbonate, sulfate, nitrate, vinyl sulfone, amide, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, alkene, ester, thioester, aryl, and / or silane.

[0034] The amino acid sequence of the hydrophilic domain is more hydrophilic than the amino acid sequence of the hydrophobic domain.

[0035] 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, and more preferably, the hydrophobic domain comprises 5 hydrophobic amino acids.

[0036] Preferably, the hydrophobic amino acid is one or more selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F) and alanine (A).

[0037] In some embodiments, the hydrophobic amino acids are one or more selected from I, V, L, A, and F.

[0038] In some embodiments, the N-terminus of the hydrophobic domain is modified with a reagent or group selected from acetyl, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, alkene, ester, thioester, aryl, and / or silane.

[0039] In some embodiments, the hydrophobic domain has an amino acid sequence selected from the following: LLLL(SEQ ID NO.:65), FIIII(SEQ ID NO.:66), IIII(SEQ ID NO.:67), IIII(SEQ ID NO.:68), ILILI(SEQ ID NO.:69), FLFLF(SEQ ID NO.:70), IVIVI(SEQ ID NO.:71), VIVIV(SEQ ID NO.:72), VLFIIV(SEQ ID NO.: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.: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).

[0040] In some embodiments, the amino acid sequence of the hydrophobic domain is more hydrophobic than the amino acid sequence of the hydrophilic domain.

[0041] In some embodiments, the self-assembling peptide further comprises a linking domain that provides a spacer region between the hydrophobic and hydrophilic domains.

[0042] In some embodiments, the linking domain comprises 2-8, preferably 4-5, amino acid residues.

[0043] In some embodiments, the linking domain comprises amino acids with small side chains, amino acids with hydroxyl groups in the side chain, and / or hydrophobic amino acids that are distant from the hydrophobic domain.

[0044] In some embodiments, the small side chain amino acids are selected from glycine (G), alanine (A), and serine (S).

[0045] In some embodiments, the amino acid having a hydroxyl group in its side chain is selected from serine (S), threonine (T), and hydroxyproline (O).

[0046] In some embodiments, the hydrophobic amino acids distal to 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.

[0047] In some embodiments, the linking domain has an amino acid sequence selected from the following: 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), where S, T and O are interchangeable.

[0048] More preferably, the linking domain has an amino acid sequence selected from: 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.:94), OGII (SEQ ID NO.:96), GOGVI (SEQ ID NO.:102) or GOGII (SEQ ID NO.:103).

[0049] In some embodiments, one or more Gs are further included between the hydrophobic domain and the linking domain to increase the flexibility and freedom of the self-assembling peptide.

[0050] In some embodiments, the self-assembling peptide is 15-50 amino acids in length, preferably 15-25 amino acids in length.

[0051] 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.

[0052] In some embodiments, the self-assembling peptide has an amino acid sequence selected from the following SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32. IIIIIGSIIGPGGDGPGGV(SEQ ID NO.1), IIIIIGSIIGPGGEGPGGV(SEQ ID NO.2), IIIIIGSIIGOGGEGPGGV(SEQ ID NO.3), IIIIGSIIGOGGEGPGGV(SEQ ID NO.4), IIIIGSIIGOGGEGPGGGV (SEQ ID NO.5)、 IIIIIGSIIGOGGEGPGV(SEQ ID NO.6)、 IIIIIGIIGOGAEGPGGV(SEQ ID NO.7)、 IIIIGSIIGOGGVGPGGV(SEQ ID NO.9)、 IIIIIIGSIGOGAEGPGGVGPGGV(SEQ ID NO.10). FLIVIGSIIGOGGEGPGGV(SEQ ID NO.11)、 FLIIAGSIIGPGGDGOGGV(SEQ ID NO.12)、 IIIIIGOGIIGPGGEGPGGE(SEQ ID NO.13)、 FIFIFGTVIGPGGEGOGGV(SEQ ID NO.14)、 IFIFIGTVIGPGGEGOGGK(SEQ ID NO.15)、 IAILIGTVIGPGGEGOGGE(SEQ ID NO.16)、 IVIVIGSIIGPGGDGPGGV(SEQ ID NO.17)、 IVIVIGSIIGOGGDGPGGV(SEQ ID NO.18)、 IVIVIGSIIGPGGEGOGGV(SEQ ID NO.19)、 FLIGHTINGIGOGGEGPGGE(SEQ ID NO.20)、 IVIVIGOGIIGOGGDGOGGV(SEQ ID NO.21)、 IVIVIGSGIIGPGGEGPGGV(SEQ ID NO.22)、 FIIIVGSIIGPGGEGPGGV(SEQ ID NO.23)、 FIIIVGSIIGPGGEGPGGE(SEQ ID NO.24)、 IIIIIGOGIIGOGGEGPGGV (SEQ ID NO.25)、 Ac-IIIIIGSIIGPGGEGOGGV(SEQ ID NO.26). FLIVIGSIIGOGAEGPGGV(SEQ ID NO.27), FLIVIGSIIGOGAEGOGGV(SEQ ID NO.28), LLLLLGSVLGPAGEGPAGE(SEQ ID NO.:29), LLLLLGPOGLGPAGEGPAGE(SEQ ID NO.:30), LLLLLGPOGVGPAGEGPAGE (SEQ ID NO.: 31), or LLLLLGPOGIGPAGEGPAGE(SEQ ID NO.:32).

[0053] The self-assembling peptides of the present invention having the above structure can form a three-dimensional network scaffold material starting from a protein-based substance or a mixed system containing a protein-based substance.

[0054] The proteinaceous substances are selected from proteins that can provide hydrogen ions under neutral physiological conditions, and preferably, the proteins are independently selected from proteins having an isoelectric point (PI) value of less than 7.0 (preferably 3.4-6.05).

[0055] In some embodiments, the protein-based agent is selected from laminin, fibronectin, fibrinogen, globulin, hemoglobin, vitronectin, transferrin, or a combination comprising one or more of the foregoing.

[0056] Mixture systems containing protein-based substances include, but are not limited to, complete cell culture media, serum-free media, animal tissue, cell preservation fluids, or other cell or drug delivery systems.

[0057] In some embodiments, the mixed system containing the protein substance is serum, plasma, cell culture medium, animal or plant tissue fluid, or animal tissue.

[0058] The present inventors have unexpectedly discovered that self-assembling peptides having the structure of the present invention can initiate and self-assemble under conditions of a protein-based substance or a mixed system containing a protein-based substance, particularly under physiological conditions, in a cell preservation culture environment and a mixed system containing a protein-based substance in a living body, preferably the human body, to form a self-assembling peptide solution system with a nano-network structure that can exert supporting and repairing functions in a solution state.

[0059] The three-dimensional network scaffold material has a nanostructure.

[0060] In a second aspect, the present invention provides a method of forming a scaffold material from the protein-responsive self-assembling peptide of the first aspect, the method comprising the step of using a protein-based material to initiate the self-assembling peptide to form the scaffold material.

[0061] Preferably, the method includes a step of mixing the self-assembling peptide with a protein-based substance or a mixed system containing a protein-based substance, or the method includes a step of injecting or implanting the self-assembling peptide into a mixed system containing the protein-based substance.

[0062] The proteinaceous substances are selected from proteins that tend to donate hydrogen ions under neutral physiological conditions, and preferably, the proteinaceous substances are independently selected from proteins having an isoelectric point (PI) value of less than 7.0 (preferably 3.4-6.05).

[0063] In some embodiments, the protein-based substance is selected from laminin, fibronectin, fibrinogen, globulin, hemoglobin, vitronectin, transferrin, or a combination comprising one or more of these, or a mixed system comprising one or more of these, including, but not limited to, complete cell culture medium, serum-free medium, animal tissue, cell preservation fluid, or other cell or drug delivery system.

[0064] In some embodiments, the mixed system containing the protein-based substance is serum, plasma, cell culture medium, animal or plant tissue fluid, animal tissue, or the like.

[0065] In some embodiments, the method comprises starting with a protein-based substance or a mixed system comprising a protein-based substance when the pH is between 6 and 10.

[0066] In some embodiments, the method comprises starting with a protein-based substance or a mixed system comprising a protein-based substance under conditions of pH 6.5-8.0, preferably pH 7.0-7.5, more preferably pH 7.2-7.4.

[0067] The self-assembling peptide can be dissolved in a neutral or alkaline solvent, and the solution can be prepared after dissolution. The solvent can include one or more aqueous solutions of sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonia water, etc., which can provide an alkaline environment.

[0068] The solvent used to dissolve the self-assembling peptide of the present invention is preferably a physiologically acceptable solution, as long as it can provide a neutral or alkaline environment. The mixed solution of the proteinaceous substance and the self-assembling peptide is adjusted to a pH of 6.5-10, preferably 6.0-8.0, more preferably 6.5-7.5, and most preferably 7.0-7.5.

[0069] The protein-responsive self-assembling peptide is initiated under conditions of a protein-based substance or a mixed system containing a protein-based substance at a temperature of 0-90°C, preferably 15-50°C, and a pH of 9 or less, and forms the scaffold material within 30 minutes, preferably within 15 minutes, and more preferably within 10 minutes.

[0070] Due to the interaction between the acidic amino acids of the self-assembling peptides of the present invention and the proteinaceous substance or the proteinaceous substance and polypeptide in a mixed system containing the proteinaceous substance, an aqueous peptide solution self-assembles into a scaffold material in the form of a hydrogel.

[0071] In particular, under physiological conditions, such as neutral physiological conditions, a protein-based substance or a mixed system containing a protein-based substance is added to provide a positive charge, thereby neutralizing the negatively charged acid ions on the self-assembling peptide molecules and reducing the repulsive force between the self-assembling peptide molecules, which then self-assemble through hydrophobic interactions and hydrogen bonds, ultimately forming a three-dimensional network nanostructure.

[0072] The advantages of the present invention are that the self-assembling peptides of the present invention can be used to prepare self-assembling peptide solutions under neutral conditions. Such self-assembling peptide solutions do not completely form a three-dimensional network scaffold material before adding a proteinaceous substance or a mixture containing a proteinaceous substance. Because of their low viscosity, operations such as mixing, extraction, and injection are extremely easy, resulting in excellent operational convenience. This is because the hydrophilic domains of self-assembling peptides contain at least one acidic amino acid, which is negatively charged under neutral conditions. Charge repulsion exists between self-assembling peptide molecules, preventing them from forming tight molecular stacks. Even if the hydrophobic effect of the hydrophobic domains provides the driving force for molecular aggregation, the electrostatic repulsion between molecules inhibits stable, regular arrangement. Furthermore, the acidic amino acids in the hydrophilic domains are hydrophilic amino acids, which tend to be exposed to the solution. The β-turn structure reduces interference from other side chains near the acidic amino acids. Therefore, the β-turn structure favors the function of the acidic amino acids. The β-turn structure makes the acidic amino acid side chains in the self-assembling peptide more active, promoting intermolecular repulsion and affecting the molecular arrangement, making it more difficult for the self-assembling peptide to form a three-dimensional network scaffold structure. Furthermore, when the self-assembling peptide molecules approach each other during movement, electrostatic forces accelerate them, increasing the kinetic energy of the entire system. Therefore, under neutral conditions, a solution containing only the self-assembling peptide molecules will be disrupted, preventing the complete formation of a three-dimensional network scaffold material.

[0073] When a positively charged protein-based substance or a mixture containing a protein-based substance is added to such a self-assembling peptide solution, the carbonyl groups on the self-assembling peptides will form electrostatic interactions with the positively charged ions / groups present. This "shields" the negative charges on the self-assembling peptide molecules, reducing intermolecular electrostatic repulsion. Hydrophobic interactions and hydrogen bonding allow the molecules to aggregate and distribute regularly, forming a three-dimensional network-like scaffold material and a macroscopic hydrogel.

[0074] The scaffold material is a three-dimensional network scaffold material and has a nanostructure.

[0075] If the hydrophobic domain is too hydrophobic, it is prone to forming associations, whereas if the hydrophobic domain is too weak, self-assembly cannot be achieved. The hydrophilic domain also allows the self-assembling peptide aqueous solution to self-assemble into a hydrogel through interaction with a proteinaceous substance or a mixed system containing a proteinaceous substance. 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 composition 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 but to form a hydrogel under initiation conditions.

[0076] The present inventors have unexpectedly discovered that self-assembling peptides having the structures described in the present invention can initiate and self-assemble to form nanonetwork structures in protein-based substances or mixed systems containing protein-based substances that are widely present in cell preservation culture environments and the human body under physiological conditions. In particular, the use of endogenous physiological substances, such as serum, plasma, cell culture media, and animal and plant tissue fluids, as mixed systems of protein-based substances significantly reduces safety risks in clinical applications, which is particularly important for drug delivery, human tissue repair, and regenerative medicine.

[0077] In some embodiments, the three-dimensional network scaffold material is in the form of a hydrogel or a dried form of a hydrogel, such as a lyophilized powder of a hydrogel.

[0078] In the hydrogel, the concentration of the protein-responsive self-assembling peptide is ≧0.1 wt %, preferably 0.3-4 wt %, more preferably 0.5-1 wt %.

[0079] The hydrogel has functions such as self-repair, and the gel state of the hydrogel is destroyed by mechanical force and recovers to the gel state after the mechanical force is removed, with the recovery time not exceeding 10 minutes, and the storage modulus of the hydrogel after recovery is at least 70% of that before destruction, preferably at least 85% of that before destruction, and more preferably 95% or more of that before destruction.

[0080] In a third aspect, the present invention provides a three-dimensional network scaffold material in the form of a hydrogel, comprising the protein-responsive self-assembling peptide of the first aspect.

[0081] In some embodiments, the three-dimensional network scaffold material is obtained by the method of the second aspect.

[0082] In some embodiments, the three-dimensional network scaffold material is in the form of a hydrogel or a dried form of a hydrogel, such as a lyophilized powder of a hydrogel.

[0083] In some embodiments, the three-dimensional network scaffold material is in the form of an injectable hydrogel.

[0084] In some embodiments, the three-dimensional network scaffold material is nanostructured.

[0085] In the hydrogel, the concentration of the protein-responsive self-assembling peptide is ≧0.1 wt %, preferably 0.3-4 wt %, more preferably 0.5-1 wt %.

[0086] The three-dimensional network scaffold material has more β-sheet structure relative to the self-assembling peptides.

[0087] In a fourth aspect, the present invention provides a composition comprising the protein-responsive self-assembling peptide of the first aspect and a protein-based substance or a mixed system comprising a protein-based substance.

[0088] The composition is in a combined or conjugated form, the latter being a combination of a self-assembling peptide and a protein-based substance or a mixed system containing a protein-based substance, each placed in a separate container.

[0089] The proteins are selected from proteins that can provide hydrogen ions under neutral physiological conditions, and preferably, the proteins are independently selected from proteins having an isoelectric point (PI) value of less than 7.0 (preferably 3.4-6.05).

[0090] In some embodiments, the protein is selected from laminin, fibronectin, fibrinogen, globulin, hemoglobin, vitronectin, transferrin, or a combination comprising one or more of these, or a mixture comprising one or more of these, including, but not limited to, complete cell culture medium, serum-free culture medium, animal tissue, cell preservation fluid, or other cell or drug delivery system.

[0091] In some embodiments, the mixed system containing the protein-based substance is serum, plasma, cell culture medium, animal or plant tissue fluid, or the like.

[0092] The present invention unexpectedly discovered that the three-dimensional network scaffold structure formed is more stable under conditions involving one or more combinations of protein-based substances, or a mixed system containing one or more of them, making such self-assembling peptides particularly suitable for in vivo applications and offering unique advantages over other self-assembling peptides in the prior art. The three-dimensional network scaffold material is a nanostructure.

[0093] At the same time, the formed hydrogel has a self-repairing function: the hydrogel's gel state is destroyed by mechanical force, and it recovers to a gel state after the mechanical force is removed, the recovery time is no longer than 10 minutes, and the storage modulus of the recovered hydrogel is at least 70% of that before destruction, preferably at least 85% of that before destruction, and more preferably 95% or more of that before destruction.

[0094] In a fifth aspect, the present invention provides applications of the protein-responsive self-assembling peptide of the first aspect, the method of the second aspect, the three-dimensional network scaffold material of the third aspect, and the composition of the fourth aspect in one or more fields selected from regenerative medicine and tissue regeneration, 2D and 3D cell culture and preservation, drug delivery, wound healing, implant materials, gene therapy, stem cell therapy, and aesthetic medicine.

[0095] The hydrogel of the present invention is safe and convenient to manufacture, does not require adjustment of the pH value, temperature, light irradiation, salt or ionic components of the system, and can be self-assembled to form a gel by some endogenous proteins commonly found in the biomedicine field, which better ensures the biocompatibility of the hydrogel of the present invention.

[0096] The protein-responsive self-assembling peptides provided by the present invention and hydrogels prepared therefrom can be used for in vitro three-dimensional culture and preservation, to construct cell models, to support cells, organs, or organoids, and can be injected into animal or human bodies for tissue repair, as wound dressings, hemostatic materials, etc., or as carriers for the sustained release of drugs or functional factors, or as cell preservation materials or in other applications in biotherapy, tissue engineering, and regenerative medicine. As described above, the protein-responsive self-assembling peptides of the present invention and hydrogels prepared therefrom are safe, convenient, and have a wide range of applications. [Brief explanation of the drawings]

[0097] [Figure 1]Figure 1A shows a macrophotograph of the protein-responsive self-assembling peptide and protein mixture solution of the present invention. Tube 1 contains a mixture of the protein-responsive self-assembling peptide and gamma globulin, while Tube 2 contains only the protein-responsive self-assembling peptide solution. The protein-responsive self-assembling peptide concentration in both centrifuge tubes is 0.3 wt%. Figure 1B shows that the self-assembling peptide can form a hydrogel using tissue fluid as an initiator, and remains in a gel state after being extruded using a syringe. [Figure 2] FIG. 2 is a TEM image of a mixture solution of the protein-responsive self-assembling peptide of the present invention and a protein. [Figure 3] FIG. 3 shows a circular dichroism spectrum of a mixture solution of the protein-responsive self-assembling peptide of the present invention and a protein. [Figure 4] Figures 4A-4B show the change in thioflavin T fluorescence in self-assembling peptide fiber hydrogels formed using fibrinogen, transferrin, and gamma globulin as initiators. [Figure 5] FIG. 5 shows the effect of different sequence structures of self-assembling peptides on the support of red blood cells forming a three-dimensional network scaffold. [Figure 6] Figure 6 shows the change in rheological properties of different self-assembling peptide sequences, in order: SEQ ID NO:7 (A), SEQ ID NO:3 (B), SEQ ID NO:2 (C), SEQ ID NO:1 (D), SEQ ID NO:5 (E), SEQ ID NO:6 (F), SEQ ID NO:4 (G), SEQ ID NO:10 (H), and SEQ ID NO:8 (I). [Figure 7] FIG. 7 shows the results of the elastic modulus of the protein-responsive self-assembling peptide of SEQ ID NO:14 and the protein mixture solution after 30 minutes, where the protein-responsive self-assembling peptide concentration was 1 wt % in both cases. [Figure 8]FIG. 8 shows the results of the elastic modulus of the protein-responsive self-assembling peptide of SEQ ID NO:18 and the protein mixture solution after 30 minutes, where the protein-responsive self-assembling peptide concentration was 1 wt % in both cases. [Figure 9] FIG. 9 is a graph showing the experimental results of shear thinning and recovery of the hydrogel of the present invention. [Figure 10] Figure 10 shows confocal laser scanning electron microscope images of hydrogels formed using the protein of the present invention to support cells, in which the concentration of the protein-responsive self-assembling peptide was 0.1 wt%. [Figure 11] FIG. 11 shows the effect of (2D) not using and (3D) using a self-assembling peptide scaffold solution on cell survival of mouse mesenchymal stem cells during refrigerated storage. [Figure 12] Figure 12 shows that 0.3 wt.%, 0.5 wt.%, and 0.7 wt.% of the self-assembling peptide of the present invention were added to a cell culture system to initiate self-assembly into a fibrous network scaffold, and liver cancer cells were cultured on it.The diameters of the cell spheres were measured on days 3, 7, 11, and 15, and characteristic cell spheres in the culture system were photographed. [Figure 13] Figure 13 shows that 0.3 wt.% of the self-assembling peptide of the present invention was added to a cell culture system to initiate self-assembly into a fiber network scaffold, and porcine muscle satellite cells were cultured on it. The diameters of the cell spheres were measured on days 1, 2, 3, and 4, respectively, and photographs of the characteristic cell spheres in the culture system were taken on days 1, 2, and 4. [Figure 14]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 aqueous solution and become uniformly suspended and dispersed after mixing with the self-assembling peptide solution. B shows that the protein-responsive self-assembling peptide mixture of microspheres is liquid. C shows that after the protein-responsive self-assembling peptide mixture of 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. [Figure 15] 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 and become uniformly suspended and dispersed after mixing with the self-assembling peptide solution. B shows that the protein-responsive self-assembling peptide mixture of microspheres is liquid. C shows that after the protein-responsive self-assembling peptide mixture of 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 OF THE INVENTION

[0098] The present invention will be further described below with reference to specific examples, which are provided by way of illustration only and do not limit the protection scope of the present invention.

[0099] The present invention provides a protein-responsive self-assembling peptide, and further provides a hydrogel prepared from the protein-responsive self-assembling peptide under conditions of a positively charged protein-based substance or a mixed system containing a protein-based substance, and the scaffold material in the form of the hydrogel is a hydrogel material having a three-dimensional network-like scaffold structure.

[0100] The protein capable of initiating the self-assembly of the protein-responsive self-assembling peptide is selected from laminin, fibronectin, fibrinogen, globulin, hemoglobin, vitronectin, transferrin, or a combination comprising one or more of these, or a mixed system comprising one or more of these, including, but not limited to, complete cell culture medium, serum-free culture medium, animal tissue, cell preservation solution, or other cell or drug delivery system.

[0101] Laminin (abbreviated as LN) is a non-collagenous glycoprotein unique to basement membranes, primarily found in the basement membrane structure. It is one of the most important components of the extracellular matrix. As a widely distributed cell surface transmembrane glycoprotein, it forms the structural basis for adhesion between epidermal cells and the basement membrane. Laminin enhances cell-cell adhesion and is involved in specific adhesion processes between cells and the extracellular matrix. It is also a signaling molecule that transmits various signals to neighboring cells through interactions with the cell surface, ensuring the connection between the epidermis and dermis and is an important substance that maintains the skin structure.

[0102] Fibronectin (abbreviated as FN) is a high-molecular-weight glycoprotein widely distributed in tissues and tissue fluids (extracellular matrix). Two subunits are cross-linked by a C-terminal disulfide bond. It is synthesized by various cell types, such as fibroblasts, astrocytes, and primary mesenchymal cells, and is deposited in various forms on the cell surface, extracellular matrix, intercellular spaces, basement membranes, and connective tissues. In biological processes, FN is involved in cell migration, adhesion, proliferation, hemostasis, tissue repair, and embryonic development. It acts as a growth factor, promoting cell proliferation, guiding epidermal cells through granulation tissue, and promoting the reconstruction of the subepidermal basement membrane and normal keratinization. Fibronectin is also involved in many pathological processes.

[0103] Fibrinogen is a glycoprotein synthesized by hepatocytes that has blood clotting function and promotes platelet aggregation. Human fibrinogen has important clinical significance. Reduction or deficiency of human fibrinogen is observed in various clinical diseases, such as severe liver disease, disseminated intravascular coagulation, postpartum hemorrhage and major surgery, and traumatic hemorrhage.

[0104] Globulin is a serum protein present in the human body. Globulin is a common protein and is basically present in all animals and plants. In the human body, globulin is divided into four types: α1, α2, β, and γ. In the present application, γ-globulin is preferred.

[0105] Hemoglobin is the main protein in red blood cells. It is a quaternary protein consisting of two alpha subunits and two beta subunits, and is the specialized protein that transports oxygen within red blood cells and gives blood its red color.

[0106] Vitronectin is a high molecular weight protein present on cell surfaces and in plasma, and is the major cell adhesion molecule, exerting structural and adhesive functions on the cell filament matrix.

[0107] The transferrin receptor is a membrane transport protein involved in the transport of iron from plasma into cells. The transferrin receptor consists of two identical 95-kD independent subunits linked by two disulfide bonds.

[0108] Among these, fibrinogen, transferrin, gamma globulin, and hemoglobin are proteins widely present in serum or plasma in the body and are important for blood coagulation, wound repair, and normal immune function. Experiments in this invention demonstrate that the self-assembling peptides of the invention can respond to these proteins and self-assemble under their protein-initiated action to form hydrogels, which can then be used as dressings for hemostasis and wound repair in vivo or in vivo. Laminin, fibronectin, and vitronectin are important components of the natural extracellular matrix and can promote cell adhesion and proliferation. The self-assembling peptides injected or implanted into the body can respond to these proteins to form hydrogels in vivo. Furthermore, transferrin can also be used as an additive component in standard serum-free media in vitro, and the self-assembling peptides can self-assemble in vitro in vitro, further expanding the scope of application of the hydrogels of the invention.

[0109] It can be understood that other protein-based substances for which detailed data are not provided in the examples of the present invention have the same or similar effect as long as they can provide a positive charge, i.e., the self-assembly of the protein-responsive self-assembling peptide into a hydrogel can be realized.

[0110] 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 associated with pH adjustment or the introduction of other exogenous substances, such as certain metal salt ions or specific proteins.

[0111] The protein-responsive self-assembling peptides of the present invention initiate self-assembly of an aqueous peptide solution into a hydrogel through the interaction between acidic amino acids and proteins. Adding a protein-based substance under neutral conditions imparts a positive charge, neutralizing the negatively charged acid ions on the self-assembling peptide molecules and reducing the repulsive forces between the self-assembling peptide molecules. The self-assembling peptide molecules then self-assemble through hydrophobic interactions and hydrogen bonding, ultimately forming a three-dimensional nano-network structure and constituting a hydrogel.

[0112] The advantages of the present invention are that the self-assembling peptides of the present invention can be used to prepare a self-assembling peptide solution under neutral conditions, and such a self-assembling peptide solution does not completely form a three-dimensional network scaffold material before the addition of a protein-based substance. Furthermore, the low viscosity of the solution maintains the ease of handling the self-assembling peptide solution before use. This is because the hydrophilic domains of self-assembling peptides contain at least one acidic amino acid, which is negatively charged under neutral conditions. Charge repulsion exists between self-assembling peptide molecules, preventing them from forming tight molecular stacks. Even though the hydrophobic effect of the hydrophobic domains drives the aggregation of self-assembling peptides, the electrostatic repulsion between molecules inhibits stable, regular arrangement. Furthermore, the acidic amino acids in the hydrophilic domains are hydrophilic amino acids, which tend to be exposed to the solution, and the β-turn structure reduces interference from other side chains nearby the acidic amino acids. Therefore, the β-turn structure favors the function of the acidic amino acids. The β-turn structure makes the acidic amino acid side chains in the self-assembling peptide more active, promoting intermolecular repulsion and affecting the molecular arrangement, making it more difficult for the self-assembling peptide to form a 3D network scaffold structure. Furthermore, as self-assembling peptide molecules approach each other during movement, electrostatic forces accelerate them, increasing the kinetic energy of the entire system. Therefore, under neutral conditions, a solution containing only self-assembling peptides results in a disordered distribution of the self-assembling peptide molecules, making it impossible to form a 3D network scaffold material. When a protein-based substance is added to such a protein-responsive self-assembling peptide solution, if positive ions / groups are present, the carbonyl groups on the protein-responsive self-assembling peptides form electrostatic interactions with them. The negative charges on the protein-responsive self-assembling peptide molecules are "shielded," reducing intermolecular electrostatic repulsion. Hydrophobic interactions and hydrogen bonding allow the molecules to aggregate and distribute regularly, forming a 3D network scaffold material.

[0113] Specifically, the hydrogen ions provided by the protein-based material of the present invention electrostatically attract the acidic amino acids (negatively charged under near-physiological and / or physiological conditions) of the self-assembling peptides, reducing and / or eliminating the mutual electrostatic repulsion between the self-assembling peptide molecules before the addition of protein, thereby providing an extremely large driving force for the self-assembling peptides to self-assemble under near-physiological and / or physiological conditions. This driving force is non-covalent crosslinking, which does not alter the physicochemical properties of the proteins themselves, allowing them to maintain and exert their original biological activity in the original system. This also demonstrates that the hydrogel of the present invention can be obtained by initiating the process under conditions of endogenous protein-based materials without the introduction of exogenous materials, and does not destroy the biological activity of the starting materials in the original system, making it a safe and highly biocompatible hydrogel. The mass fraction ratio of the protein-based material to the self-assembling peptide in the hydrogel of the present invention is (1-100):(100:1).

[0114] The concentration of the self-assembling peptide in the hydrogel of the present invention is preferably 0.1 wt% or more, preferably 0.3-4 wt%, and more preferably 0.5-1 wt%. The self-assembling peptide of the present invention responds quickly to proteinaceous substances, and within a short period of time, preferably within 30 minutes, preferably within 15 minutes, or even within 10 minutes, after contact between the self-assembling peptide and the proteinaceous substance, the two self-assemble into a hydrogel through non-covalent crosslinking, i.e., gelation is achieved.

[0115] The protein-responsive self-assembling peptides of the present invention not only have the advantage of rapidly assembling to form hydrogels in a short time, but also have the property of rapidly returning to their original state after shear thinning and removal of external force. This indicates that the application of a strong external mechanical force ruptures some of the non-covalent bonds within the hydrogel, macroscopically destroying the gel state and converting it to a solution state. After the mechanical force is removed, these ruptured non-covalent bonds reconnect, and the macroscopically destroyed gel self-repairs to a state close to its original gel state. Furthermore, the hydrogel recovery time does not exceed 10 minutes, and after recovery, the storage modulus is at least 70% of its original value, and can even reach 85% or even 95% or more. Furthermore, the hydrogels of the present invention retain their self-repairing ability even after multiple shear thinnings. This means that shear thinning only temporarily destroys the three-dimensional network structure within the hydrogel, allowing it to potentially form a gel in situ after injection. Shear thinning can be performed using mechanical forces that can apply shear or shear stress to the hydrogel, such as pipetting, centrifugation, shaking, injection, spraying, filtration, etc. Furthermore, the hydrogel of the present invention has the advantage that the self-repair process after shear thinning can be terminated by dilution. That is, after shear thinning by mechanical force, the hydrogel cannot recover to a gel state by diluting with a solvent, but becomes a liquid, which is advantageous for separating substances carried by the hydrogel. For example, after cells are cultured in the hydrogel and then stored and transported, they can be separated from the hydrogel by shear thinning and sufficient dilution, and then harvested.

[0116] The hydrogels of the present invention minimize the need to alter the conventional application environment during use. They self-assemble in response to protein-based materials in an in vivo environment without the introduction of exogenous substances, making them suitable for a variety of applications, including tissue repair, in vitro 3D cell culture, cell and virus storage, wound dressing fabrication, and the preparation of scaffolds required for tissue engineering. They also support injection, which is advantageous for in vitro use in cell culture fluid exchange and cell isolation, and for in vivo use in achieving in-situ repair and in vivo drug delivery. In vitro 3D cell culture does not require the addition of any other components beyond the complete cell culture medium. For some immunological applications, protein-based materials within the application scenario may be selected as the component that initiates the self-assembly of self-assembling peptides to avoid experimental interference due to the introduction of other proteins. At the same time, the hydrogels of the present invention do not require changes in the temperature and pH of the entire system during the self-assembly process and / or after gel formation, providing significant security for biomedicine applications and enabling broad applications in the biomedicine field.

[0117] In some embodiments, the hydrogels of the present invention are used for three-dimensional cell culture. The self-assembling peptide solution of the present invention can be directly mixed with a complete cell culture medium containing cells and then transferred to cultureware such as a culture dish, culture bottle, or cell culture well plate to achieve three-dimensional cell culture. The external environmental conditions and culture medium required for three-dimensional culture are consistent with those of conventional two-dimensional cell culture, eliminating the need for expensive equipment. The serum in the complete cell culture medium contains various proteins, such as vitronectin and gamma globulin, which can induce the self-assembling peptides of the present invention to self-assemble into hydrogels, thereby supporting cells and promoting cell proliferation. The hydrogels of the present invention are suitable for three-dimensional culture of various types of cells.

[0118] Terminology Hydrogels are hydrophilic polymeric materials that can form three-dimensional network structures through chemical or physical crosslinking. Hydrogel materials can be of natural or synthetic origin. Natural polymers such as chitosan, alginate, hyaluronic acid (HA), collagen, and gelatin have the advantages of being biodegradable and possessing integrin binding sites, but they are immunogenic. Synthetic polymers such as polyethylene glycol (PEG), polyacrylamide (PAM), polyvinyl alcohol (PVA), and polymethyl methacrylate (PMMA) have advantages such as strong mechanical properties, customization, and low immunogenicity, but lack inherent biological function and require significant post-processing to initiate the desired response in vivo.

[0119] The hydrogel has the following properties: 1. Good biological relevance: The polymer contains many hydrophilic groups, and can absorb water several dozen times its own weight. It also has the property of swelling in water without dissolving, giving it good water retention capacity. 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 advantageous for cell growth and proliferation. 3. Biodegradability: Some natural polymer materials are biodegradable, avoiding secondary damage caused by removal of the implant. Due to these unique advantages, hydrogels are playing a major role in biomedical materials.

[0120] In the present invention, a peptide having both a hydrophilic surface and a hydrophobic surface structure self-assembles under the conditions described in the present invention, particularly under physiological conditions, and thereby encapsulates water to form a hydrogel.

[0121] Self-assembling peptides (SAS) are short amino acid chains containing polar domains. When dissolved in neutral solvents and physiological salt concentrations, these self-assembling peptides spontaneously organize into hierarchical nanostructures through hydrogen bonding, ionic bonding, hydrophobic interactions, or van der Waals forces. Materials derived from these components have the advantages of being nontoxic, 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 be further functionalized with cell-interacting peptide domains or the attachment of cytokines and growth factors, enabling them to be used to design biologically relevant culture environments and improve the control of proliferating cell populations.

[0122] Self-assembling peptides can be used as drug carriers in wound healing hydrogels, as polypeptide nanofibers in cancer treatment, and for sustained release of small molecules, growth factors, and monoclonal antibodies. For example, self-assembling peptides can be used to promote angiogenesis in regenerating tissue and to study the repair of skin wound surfaces using polypeptide scaffolds. However, the use of self-assembling peptides in injectable hydrogel therapy is still in its infancy.

[0123] Self-assembling peptides contain alternating hydrophilic and hydrophobic amino acid residues, allowing them to retain large amounts of water and form hydrogels. The hydrophilic residue side chains can directly interact with water, and water molecules form inclusion compounds around the hydrophobic residue side chains. The number and ratio of hydrophobic and hydrophilic residues in a self-assembling peptide must be carefully designed. If there are too many hydrophobic residues, the self-assembling peptide will not dissolve in water and will precipitate from it. On the other hand, if there are too many hydrophilic residues, the self-assembling peptide will be highly water-soluble and will not form a hydrogel. Furthermore, the self-assembly of peptide molecules must be precisely and skillfully induced to form regular nanostructured materials, such as nanofibers, nanotubes, and nanovesicles.

[0124] "Amino acid" includes naturally occurring and non-naturally occurring amino acids, e.g., D-natural amino acids, β and γ derivatives. In accordance with standard terminology, amino acid residue sequences may include, for example, alanine (Ala, A), arginine (Arg, R), aspartamide (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 They can be named with a three-letter or one-letter code, such as (Lysine, L), methionine (Met, M), phenylalanine (Alanine, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V), selenocysteine ​​(Sec, U), and hydroxyproline (Hyp, O).

[0125] In the present invention, a "peptide" is an amino acid chain. In particular, a peptide is 2 to 40 amino acids in length.

[0126] In the present invention, "self-assembly" refers to the aggregation of self-assembling peptides under normal environmental conditions to form a regular structure.

[0127] In the present invention, "β-sheet" refers to a relatively extended, periodically sheeted, zigzag main chain conformation in a polypeptide chain, which is arranged in a parallel or antiparallel manner to form a β-sheet (strand). The parallel or antiparallel conformation is determined based on the direction in which the peptides are arranged from the N- to C-terminus. A parallel arrangement refers to peptide chains arranged in the N- to C-terminus direction. An antiparallel arrangement refers to peptide chains arranged in opposite directions (i.e., a first peptide chain is arranged from the N- to C-terminus, and an opposing second peptide chain is arranged from the C- to N-terminus). A parallel arrangement may include crossing of the peptide ends caused by translation of the peptide. At least half of the peptide length is involved in the interpeptide interaction force. In an antiparallel arrangement, polypeptides are usually arranged in a line to provide flush ends. This is a typical end-to-end complementary peptide.

[0128] A β-turn is an irregular secondary structure in proteins that causes a change in the direction of the polypeptide chain. A β-turn often occurs when a peptide chain folds back 180°. A β-turn consists of 3-5 amino acid residues, the second of which is proline (P) or hydroxyproline (O). A β-turn motif is typically a turn structure stabilized by a hydrogen bond 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-bends, reverse bends, or β-loops, are used to connect β-chains.

[0129] "Hydrophobic" in the present invention refers to a property of tending to repel water or not dissolving in water at all.

[0130] In the present invention, "hydrophilic" refers to having the property of a polar group that easily absorbs moisture and easily interacts strongly with water.

[0131] Hydrophilic amino acids, or polar amino acids, have a certain affinity for water molecules because their R groups are polar and can generally form hydrogen bonds with water molecules. Hydrophilic amino acids include S, T, Y, C, U, N, Q, D, E, O, R, K, and H.

[0132] Hydrophobic amino acids, or nonpolar amino acids, whose R groups are nonpolar, have little or no affinity for water molecules but a high affinity for lipid-soluble substances. These include G, A, V, L, I, P, M, F, and W.

[0133] "Nanostructure" refers to a structure having a nanometer size. Nanostructures can be any shape in one, two, or three spatial dimensions, including nanofilms, nanofibers, nanorods, nanowires, nanofiber networks, nanoballs, nanospirals, and some mixtures thereof. The surface of a nanostructure has a one-dimensional structure at the nano level, i.e., the thickness of the surface of the object is 0.1-100 nm. Nanotubes have two dimensions of nanosize, with a diameter of 0.1-100 nm and a length that can be greater. The nanosize of spherical nanoparticles has three dimensions, i.e., the size of each spatial dimension of the nanoparticle is 0.1-100 nm.

[0134] When a protein solution is at a certain pH, the protein has an equal tendency to dissociate into positive and negative ions, i.e., zwitterions, with a net charge of zero; the pH of the solution at this point is called the isoelectric point (pI) of the protein.

[0135] Circular dichroism spectroscopy is the most widely applied method to measure the secondary structure of proteins and monitor the conformational changes of protein molecules due to external conditions. It is a fast, simple and more accurate method for studying protein conformation because it detects liquids and the obtained results are closer to the secondary structure of proteins in real physiological environments.

[0136] In the present invention, the terms "response" and "initiation" both refer to the formation of a hydrogel by a protein-responsive self-assembling peptide in response to a different proteinaceous substance, or the formation of a hydrogel by initiating the protein-responsive self-assembling peptide with a proteinaceous substance, and have the same meaning.

[0137] The present invention will be further described below with reference to specific examples. Because the protein-responsive self-assembling peptides of the present invention share the same principle of forming hydrogels in response to different proteins, several examples provide verification data for the properties and functions of the hydrogels of the present invention, using different protein-responsive self-assembling peptide sequences as examples. It is understood that other self-assembling peptides that are compatible with the molecular structure of the self-assembling peptides of the present invention, for which detailed data is not provided in the present invention, will have the same or similar effects. The amino acids referred to in the present invention are represented by single-alphabetical abbreviations, in the format generally accepted in the art.

[0138] Example 1 Synthesis of self-assembling peptides The self-assembling peptides of the present invention were synthesized using standard solid-phase polypeptide synthesis methods. The amino acid sequences of the self-assembling peptides are shown in SEQ ID NOs. 1-32 in the Sequence Listing. High-performance liquid chromatography and mass spectrometry confirmed that they were the desired self-assembling peptides.

[0139] Example 2 Preparation of self-assembling peptides Preparation of a 2 wt% self-assembling peptide solution: Ultrapure water is added to an appropriate amount of the powder of the self-assembling peptides of SEQ ID NOs. 1-32 obtained in Example 1. A small amount of alkaline solution is used to completely dissolve the self-assembling peptide. After dissolution, the pH of the solution is adjusted to neutral (e.g., about 6.5 to about 8, about 7.0-7.5, or even about 7.0-7.2) using trace amounts of dilute hydrochloric acid and a weak alkaline solution. 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 peptides of SEQ ID NOs. 1-32. This solution is stored at 4°C for use. Upon application, the self-assembling peptide mother solution is diluted with PBS buffer or ultrapure water to obtain a solution of the self-assembling peptides of SEQ ID NOs. 1-32 at the desired concentration. For example, an appropriate amount of 2 wt% self-assembling peptide mother solution is diluted 10-fold with PBS buffer to obtain a 0.2 wt% self-assembling peptide solution.

[0140] Example 3 Preparation of Hydrogel The hydrogel of the present invention can be obtained by uniformly mixing the self-assembling peptide solutions shown in SEQ ID NOs. 1-32 obtained in Example 2 with a protein solution and adjusting the pH of the mixed solution to neutral (e.g., about 6 to about 8, preferably about 6.5-7.5, more preferably about 7-7.5). The protein is dissolved in a phosphate buffer (neutral pH) or a basal cell culture medium to obtain a protein solution. The mass fraction ratio of the self-assembling peptide to the protein in the mixed solution was (1-100):(100-1). Using this method, hydrogels of the present invention with any desired concentration of the self-assembling peptide can be prepared.

[0141] Proteins used were laminin (PI approximately 4.94-6.05), fibronectin (PI approximately 5.25), fibrinogen (PI approximately 4.8), transferrin (PI approximately 3.4-3.5), gamma globulin (PI approximately 5.8), vitronectin (PI approximately 5.47), collagen (PI approximately 7.5-7.8), and hemoglobin (PI approximately 7.23). 1 wt% solutions of laminin, fibronectin, fibrinogen, transferrin, gamma globulin, vitronectin, hemoglobin, and human collagen were prepared in phosphate buffer at a neutral pH. These solutions were then uniformly mixed with the 1 wt% self-assembling peptide mother solution obtained in Example 2 at a 1:1 volume ratio to obtain the mixture systems of the Examples and Comparative Examples. Table 1 shows the response of each protein to the self-assembling peptides indicated by their respective sequences.

[0142] [Table 1]

[0143] The results in Table 1 indicate that the self-assembling peptides of the present invention can form hydrogels in response to proteins with an isoelectric point of less than 7.0 (e.g., laminin, fibronectin, fibrinogen, transferrin, gamma globulin, and vitronectin). By comparison, it was found that the self-assembling peptides of the present invention can only form weak hydrogels (e.g., hemoglobin) or cannot initiate hydrogel formation (e.g., human collagen) when initiated by proteins with an isoelectric point higher than neutral.

[0144] The results in Table 1 demonstrate that the self-assembling peptides of the present invention can form hydrogels in response to compositions and mixtures containing proteins with an isoelectric point of less than 7.0, such as natural serum and plasma.

[0145] Figure 1A shows the hydrogel obtained by mixing SEQ ID NO. 24 (FIIIVGSIIGPGGEGPGGE) as a self-assembling peptide and gamma globulin as a protein. The self-assembling peptide solution is liquid, and after rotating the centrifuge tube 90°, it flows until the liquid surface becomes parallel to the ground (tube 2 in Figure 1A), remaining in a solution state. In the presence of gamma globulin, the self-assembling peptide self-assembles into a hydrogel in response to gamma globulin (tube 1 in Figure 1A). Rotating the centrifuge tube 90° reduces the flow rate as the hydrogel is formed, but the liquid surface remains unchanged and perpendicular to the ground. Figure 1B shows that the self-assembling peptide can form a hydrogel using tissue fluid as an initiator, and remains in a gel state even after being extruded from the syringe. The self-assembling peptides shown in SEQ ID NOs. 1-32 have the same or similar effects on hydrogels formed (labeled with √ or ~) in response to other proteins (e.g., laminin, fibronectin, fibrinogen, transferrin, gamma globulin, vitronectin, or a mixture thereof).

[0146] Taking the self-assembling peptide represented by the amino acid sequence LLLLLGSVLGPAGEGPAGE (SEQ ID NO: 29) as an example, a 2% concentration of the self-assembling peptide was mixed with an equal volume of human tissue fluid to rapidly form a hydrogel that could be drawn up with a syringe and maintained in a gel state even after being extruded by injection. Peptide hydrogel materials formed in response to other types of starting substances also have equivalent or similar effects.

[0147] Example 4 Structural Characterization of Hydrogel Materials Experiment 1: Transmission Electron Microscope (TEM) Experimental method: Preparation of self-assembling peptide solution used in TEM experiments: The 0.2 wt % self-assembling peptide solution obtained in Example 2 was diluted with ultrapure water until the self-assembling peptide concentration reached 0.002 wt %.

[0148] Preparation of protein solutions used in TEM experiments: Laminin, fibronectin, fibrinogen, transferrin, gamma globulin, and vitronectin were each added to ultrapure water to a protein concentration of 0.002 wt%.

[0149] Preparation of self-assembling peptide samples: The 0.2 wt% self-assembling peptide solution obtained in Example 2 was diluted to 0.002 wt% with ultrapure water and incubated at 37°C for 1 hour. 10 μL was then placed on a 300-mesh carbon-supported copper grid (purchased from Beijing Xinxing Bairui Technology Co., Ltd.) and allowed to dry at room temperature. After drying, 10 μL of 2 wt% phosphotungstic acid negative stain was added to the copper grid with the sample. After staining for 60 seconds, excess stain was removed and this staining step was repeated three times. After staining was complete, the copper grid was allowed to dry at room temperature.

[0150] Preparation of the hydrogel samples of the present invention: 0.002 wt% self-assembling peptide solution was uniformly mixed with 0.002 wt% protein solution at a 1:1 volume ratio and incubated at 37°C for 1 hour. 10 μL of each solution was then 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. As the mixture stood, the water evaporated, gradually concentrating the self-assembling peptide and protein to form a hydrogel. After allowing to dry completely, 10 μL of 2 wt% phosphotungstic acid negative stain was applied to the copper grid with the sample. After staining for 60 seconds, excess stain was blotted off. This staining step was repeated three times. After staining was complete, the copper grid was allowed to dry at room temperature.

[0151] All samples were observed and photographed using a Talos G2 200X transmission electron microscope. Figure 2 shows the results of imaging the hydrogels formed by the self-assembling peptide shown in SEQ ID NO. 13 (IIIIIIGOGIIGPGGEGPGGE) with fibrinogen, transferrin, and gamma globulin, respectively.

[0152] When self-assembling peptide molecules are present alone (see the control group), they can assemble into fibers, but the fiber lengths are not uniform, the distribution is uneven, the overall length is short, and the fibers are rarely entangled or bundled. In contrast, when the concentration of self-assembling peptide is the same, in response to protein (see the fibrinogen-initiated group, transferrin-initiated group, and gamma globulin-initiated group), the number and length of fibers formed by the self-assembling peptide molecules are significantly increased, the degree of crimping is reduced, and the fiber arrangement pattern is also changed, resulting in more uniform fiber distribution, cross-linking and aggregation between fibers into bundles, and the fiber bundles are entangled with each other, forming a dense, uniformly voided three-dimensional network structure. This phenomenon indicates that protein initiation leads to more stable and regular self-assembly of the self-assembling peptides, thereby forming a denser, more stable, stronger, and more uniformly voided three-dimensional nanofiber network structure that can efficiently retain moisture and provide good support. This structure is advantageous in supporting cells during cell culture and in constructing a three-dimensional growth space for cells.

[0153] Experiments on self-assembling peptides designated by other sequence numbers gave results similar to those of this example, and therefore detailed explanations will be omitted here.

[0154] Experiment 2: Circular dichroism (CD) analysis 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 polypeptide concentration of 0.02 wt%.

[0155] Preparation of protein solutions for CD detection: Laminin, fibronectin, fibrinogen, transferrin, gamma globulin, and vitronectin were each added to ultrapure water to a protein concentration of 0.02 wt%.

[0156] Preparation of self-assembling peptide sample: The 0.2 wt % self-assembling peptide solution obtained in Example 2 was diluted with PBS buffer to a polypeptide concentration of 0.01 wt %, and incubated at 37° C. for 1 hour.

[0157] Preparation of the hydrogel sample of the present invention: 0.02 wt% self-assembling peptide solution was uniformly mixed with 0.02 wt% protein solution in a 1:1 volume ratio and incubated at 37°C for 1 hour to obtain a mixed system with a final self-assembling peptide concentration of 0.01 wt%. Because the self-assembling peptide / protein hydrogel is a swelling system, the concentration of the self-assembling peptide in this experiment was less than 0.1 wt%, but these results verify the interaction between the self-assembling peptide and the protein.

[0158] Each sample was placed in a 1 mm rectangular quartz cuvette (400 μL) and subjected to circular dichroism detection in the 190-260 nm wavelength range at room temperature using a MOS-450 / AF-CD spectrometer (Bio-Logic, Claix, France) at a resolution of 0.5 nm and a scanning speed of 0.5 nm / s. Each detection set was performed in triplicate after background subtraction. The results are shown in Figure 3 for the hydrogels formed by the self-assembling peptide SEQ ID NO. 13 (IIIIIGOGIIGPGGEGPGGE) with fibrinogen, transferrin, and gamma globulin, respectively.

[0159] Circular dichroism detection results showed that the response of the self-assembling peptide to proteins manifests as changes in secondary structure. Figure 3 shows that when the self-assembling peptide is present alone in a neutral environment (see the control group), the secondary structure of the nanofibers assembled by it is primarily β-sheet. In the systems where proteins are introduced, different degrees of rightward shifts are observed in the positive peak around 200 nm in the circular dichroism spectra. This indicates that the response to proteins increases the β-turns in the secondary structure of the nanofibers assembled by the self-assembling peptide, forming more β-sheet structures, providing a favorable basis for making the fiber structure more regular and ordered, compared to when the self-assembling peptide is present alone and not initiated. Furthermore, in the protein-added groups (see fibrinogen-initiated group, transferrin-initiated group, and gamma globulin-initiated group), a negative peak appeared around 192 nm, indicating the expression in the secondary fiber structure of a characteristic polymerized structure formed by the involvement of proline or hydroxyproline in the assembly of the self-assembling peptide. This indicates that the response to these three substances results in more diverse secondary structures of fibers assembled by the self-assembling peptides of the present invention, and that the increased secondary structure promotes the stability of the fiber structure. As can be seen from this experiment, while the self-assembling peptide itself can assemble into nanofibers with specific secondary structures, the response to proteins causes the polypeptides or their derivatives to assemble into nanofibers with more complex and ordered structures, thereby exhibiting a macroscopic three-dimensional network-like structure with tightly cross-linked fibers and uniform void spaces, as seen in Experiment 1.

[0160] Experiment 3: Thioflavin T fluorescence experiment Experimental Method: Thioflavin T binds to the β-sheet structure of self-assembling peptides, enhancing their fluorescence intensity. In this experiment, we investigated the secondary structural changes during the self-assembly process of self-assembling peptides by detecting the fluorescence change of Thioflavin T using SEQ ID NO: 13 (IIIIIIGOGIIGPGGEGPGGE). 800 μM (0.148 wt%), 400 μM (0.072 wt%), and 200 μM (0.036 wt%) self-assembling peptide solutions were mixed with 0.072 wt% protein-based initiator at a volume ratio of 1:1. Ultrapure water was used instead of the protein-based initiator as a control. After allowing to stand for 15 minutes, the mixture was mixed with an equal volume of 100 μM Thioflavin T solution. Fluorescence emission measurements were performed in the 450-550 nm range, and five spectra were acquired for each sample. The excitation wavelength was 442 nm, with excitation and emission slits of 5 nm and 2.5 nm, respectively.

[0161] Experimental Results: The CD spectra were verified using thioflavin T (ThT), which stains peptide fibers rich in β-sheet structure. The presence of β-sheets was confirmed and correlated with peptide concentration. The self-assembling peptide gels contained β-sheets, and their content was positively correlated with the self-assembling peptide concentration, demonstrating that the self-assembling peptides exhibited concentration-dependent self-assembling (Figure 4A). This is because an increase in the amount of peptide monomers facilitates intermolecular contact, improving the degree of assembly. The β-sheet content of the gels formed after the addition of a protein-based initiator was significantly higher than that of the gels formed after the addition of a protein-based initiator (Figure 4B). CD and ThT staining data indicated that, under neutral pH conditions, the protein initiator activated the self-assembling peptides, and the resulting supramolecular polymers exhibited a typical β-sheet structure.

[0162] Example 5: Verification of the influence of the amino acid sequence structure and starting components on the physical properties and functionality of the peptide self-assembling material of the present invention, and the mechanism of self-assembly Experiment 1: Verification of support for red blood cells by different arrangements under liquid conditions In this experiment, the influence of amino acid sequence on cell support was examined using red blood cells as an example, and in particular the influence of hydroxyproline and alanine in the amino acid sequence on cell support was examined.

[0163] Experimental method: SEQ ID NO:1 (IIIIIGSIIGPGGDGPGGV), SEQ ID NO:2 (IIIIIGSIIGPGGEGPGGV), SEQ ID NO:3 (IIIIIGSIIGOGGEGPGGV), and SEQ ID NO:7 (IIIIIGSIIGOGAEGPGGV) self-assembling peptide mother solutions were prepared in PBS buffer at a concentration of 0.1 wt% and sterilized at high temperature.

[0164] In a 2 mL transparent glass bottle, 2 mL of the self-assembling peptide solution (containing fibronectin at a final concentration of 0.1 wt%) containing the four materials (0.1 wt%, 0.05 wt%, and 0.01 wt%) was placed. A blank group (containing fibronectin at 0.1 wt% but no self-assembling peptide solution) was also placed. 5 × 10 9 Add erythrocyte mother solution to a final concentration of 1 x 10 8 The mixture was then pipetted evenly. Images were taken every four hours to compare the supportive effects of different materials on red blood cells. Because the hydrogel formed by the self-assembling peptides of the present invention is a water-swellable system, the low-concentration polypeptide network scaffold remains in solution, yet the supportive ability of the polypeptide hydrogel was still demonstrated.

[0165] Experimental conclusion: Among self-assembling peptides with similar amino acid sequences, hydroxyproline and alanine have a significant effect on the cell-supporting capacity of hydrogels or scaffold solutions formed by swelling of the hydrogels. As shown in Figure 5, under high concentration conditions (0.1 wt% and 0.05 wt%), all four self-assembling peptide solutions exhibited cell-supporting capacity of 1×10 8It can effectively support red blood cells. However, under low-concentration conditions (0.01 wt%), hydroxyproline, alanine, and glutamic acid in the hydrophilic domain also showed a significant impact on the red blood cell support ability. The four self-assembling peptide solutions showed significant precipitation at 8 hours, 24 hours, 32 hours, and 48 hours respectively. The support abilities of the four self-assembling peptides, from weak to strong, are in the order of SEQ ID NO:1 (IIIIIGSIIGPGGDGPGGV) < SEQ ID NO:2 (IIIIIGSIIGPGGEGPGGV) < SEQ ID NO:3 (IIIIIGSIIGOGGEGPGGV) < SEQ ID NO:7 (IIIIIGSIIGOGAEGPGGV). The scaffold network formed by the fibronectin response showed that glutamic acid in the hydrophilic region of SEQ ID NO:2, glutamic acid and hydroxyproline in the hydrophilic region of SEQ ID NO:3, and glutamic acid, hydroxyproline, and alanine in the hydrophilic region of SEQ ID NO:7 successively showed an enhancing effect on the cell support ability with respect to SEQ ID NO:1. Hydroxyproline is abundant in the collagen tissues of animals, and the hydrogen bonds formed by the hydroxy groups in it can enhance the elasticity and support force of protein substrates. In this patent, by introducing hydroxyproline, the stability of the β-sheet formed by the self-assembly of self-assembling peptides is significantly enhanced through the strengthened hydrogen bond network between self-assembling peptides, the stability of the self-assembling scaffold is improved, and the cell support function can be strengthened. At the same time, the β-sheet structure in the hydrophilic region can enhance the hydrophobicity of the amino acid side chains and improve the hydrophobic interaction between polypeptides without destroying the β-turn structure of the hydrophilic region by substituting glycine with a small number of alanines and substituting aspartic acid with glutamic acid, thereby similarly improving the stability of the self-assembling material, and thus making the support of the scaffold material for cells stronger.

[0166] Experiment 2 Rheology Experiment, Influence of Secondary Structure and Amino Acid Sequence of Self-Assembling Peptides Experimental method: Preparation of self-assembling peptide material sample: 2 wt% self-assembling peptide solutions obtained in Example 2 were used: SEQ ID NO:1 (IIIIIGSIIGPGGDGPGGV), SEQ ID NO:2 (IIIIIGSIIGPGGEGPGGV), SEQ ID NO:3 (IIIIIGSIIGOGGEGPGGV), SEQ ID NO:4 (IIIIGSIIGOGGEGPGGV), SEQ ID NO:5 (IIIIIGSIIGOGGEGPGGGV), SEQ ID NO:6 (IIIIIGSIIGOGGEGPGV), SEQ ID NO:7 (IIIIIGSIIGOGAEGPGGV), SEQ ID NO:8 (IIIIIGSIIOGGAEGPGGV), SEQ ID NO:9 (IIIIIGSIIGOGGVGPGGV), SEQ ID NO:10 (IIIIIIGSIIGOGAEGPGGVGPG GV).

[0167] Preparation of hydrogel samples formed from self-assembling peptide solutions in a cell-complete medium response: The 2 wt% self-assembling peptide material mother solution obtained in Example 2 was diluted with phosphate buffer, and calf serum was added to obtain a mixed solution with a final self-assembling peptide concentration of 0.5%, a final serum concentration of 10 v / v%, and a neutral pH.

[0168] The storage modulus (G') of the mixed solutions was measured on a 20 mm plate using a MARS 60 rheometer. To determine the formation rate of the three-dimensional nanomatrix, the solutions were prepared and immediately placed on the plate for testing. A 500 mm gap was used, and mineral oil was added to the gap to prevent sample dehydration, and data collection was initiated. Dynamic time scan experiments (DTS) were performed to monitor the change in storage modulus (G') with time (at a frequency of 1 Hz and a strain of 1%) and continued for 2000 minutes.

[0169] Experimental Results: The mechanical strength of the peptide hydrogel of the present invention can effectively support and encapsulate cells, functional molecules, and drugs, making it advantageous for three-dimensional cell culture, tissue repair, and sustained drug release. Experiments demonstrated that by mixing a mixed peptide solution with serum, the self-assembling peptides self-assembled in response to the starting material, resulting in a hydrogel material with a storage modulus greater than 10. The procedure is simple and the gelation time is short. Comparison of different sequences revealed that SEQ ID NO:7 (IIIIIGSIIGOGAEGPGGV) > SEQ ID NO:3 (IIIIIGSIIGOGGEGPGV) > SEQ ID NO:2 (IIIIIGSIIGPGGEGPGGV) ≥ SEQ ID NO:1 (IIIIIGSIIGPGGDGPGGV) (see Figure 6). This experimental phenomenon is consistent with the results of Experiment 3, namely, the substitution of hydroxyproline and alanine (A) for glycine (G) in the hydrophilic region can enhance the mechanical strength of the hydrogel formed by the present invention, thereby enhancing the support function of the scaffold material. Similarly, comparison of SEQ ID NO:5 (IIIIIGSIIGOGGEGPGGGV) and SEQ ID NO:6 (IIIIIGSIIGOGGEGPGV) revealed that the self-assembling peptide can still form a weakly elastic hydrogel structure when the number of β-turn-forming amino acids is six (SEQ ID NO:5) and three (SEQ ID NO:6), when the number of increased β-turn structures is three consecutively (SEQ ID NO:10), or when the number of hydrophobic amino acids is four (SEQ ID NO:4). In Figure 6, A corresponds to sequence SEQ ID NO:7, B corresponds to sequence SEQ ID NO:3, C corresponds to sequence SEQ ID NO:2, D corresponds to sequence SEQ ID NO:1, E corresponds to sequence SEQ ID NO:5, F corresponds to sequence SEQ ID NO:6, G corresponds to sequence SEQ ID NO:4, H corresponds to sequence SEQ ID NO:10, and I corresponds to sequence SEQ ID NO:8.

[0170] In SEQ ID NO:8, the amino acids GO at positions 10-11 in SEQ ID NO:7 were changed to OG, completely destroying the conditions for the formation of the first β-turn in SEQ ID NO:7, resulting in the formation of SEQ ID NO:8. As a result, we discovered that SEQ ID NO:8 completely lost its self-assembling response to serum. This phenomenon further demonstrated that two consecutive β-turns are the most fundamental requirement for the self-assembling peptides of the present invention to have self-assembly capabilities. Comparing SEQ ID NO:9 (IIIIIGSIIGOGGVGPGGV) with SEQ ID NO:3 (IIIIIGSIIGOGGEGPGGV) revealed that without an acidic amino acid at the end of the β-turn, the aqueous solution of the self-assembling peptide did not respond to serum but could form a hydrogel in a neutral aqueous solution. This further verified that an acidic amino acid at the end of the β-turn in at least one hydrophilic region of the self-assembling peptide is a prerequisite for the self-assembling peptide of the present invention to respond to the starting components of the self-assembling materials.

[0171] Example 6 Measurement of the mechanical properties of hydrogels Experiment 1: Measurement of the mechanical strength of hydrogels (part 1) Experimental method: Preparation of self-assembling peptide solution used in dynamic rheology test: The self-assembling peptide mother solution prepared in Example 2 with a self-assembling peptide concentration of 2 wt % was taken.

[0172] Preparation of protein solutions for dynamic rheological testing: Laminin solutions, fibronectin solutions, fibrinogen solutions, vitronectin solutions, gamma globulin solutions, transferrin solutions, hemoglobin solutions, and human collagen solutions were prepared using ultrapure water with a protein concentration of 2 wt%.

[0173] Preparation of self-assembling peptide sample: The 2 wt % self-assembling peptide mother solution from Example 2 was diluted to 1 wt % with ultrapure water.

[0174] Preparation of hydrogel samples: 2 wt% self-assembling peptide mother solution was uniformly mixed with the above 2 wt% laminin, fibronectin, fibrinogen, vitronectin, gamma globulin, transferrin, hemoglobin, and human collagen solutions at a 1:1 volume ratio to obtain a mixed system with a final self-assembling peptide concentration of 1 wt%.

[0175] To simultaneously measure the gel formation rate and strength of the mixed systems, 200 μL of each sample was immediately added to a 20 mm diameter panel of a MARS 60 rheometer after preparation. After a 30-minute time scan, the final elastic modulus was recorded. The rotor-to-panel gap was 500 μm, the shear strain was 1%, and the frequency was 1 Hz. The entire test was completed at 37°C. The results are shown in Figure 7 for mixed systems formed with the self-assembling peptide shown in SEQ ID NO. 14 (FIFIFGTVIGPGGEGOGGV) and laminin, fibronectin, fibrinogen, vitronectin, transferrin, gamma globulin, hemoglobin, and human collagen, respectively.

[0176] As a result, for each sample containing 1 wt% self-assembling peptide, when the self-assembling peptide was present alone, the storage modulus was higher than the loss modulus, but not significantly different, and both were below 2 Pa (not shown), indicating a liquid rather than a solid state at the macroscopic level. However, when protein was present in the sample, the mixed self-assembling peptide material of the present invention showed a rapid increase in storage modulus (G') within 5 min, which was significantly higher than the loss modulus (G"), forming a high-strength hydrogel with a modulus exceeding 100 Pa within 30 min (Figure 7 shows G' and G" at 30 min). This indicates that the self-assembling peptide molecules of the present invention can rapidly assemble in the presence of positively charged natural substances (particularly proteins with an isoelectric point PI below 7.0) to form hydrogels with a certain mechanical strength. Due to its high isoelectric point, hemoglobin can only form weak gels, and the collagen mixed solution cannot form a gel at all.

[0177] Experiment 2: Measurement of the mechanical strength of the hydrogel of the present invention (part 2) Experimental method: As in Experiment 3, the results are shown in Figure 8 for a mixed system consisting of the self-assembling peptide shown in SEQ ID NO. 18 (IVIVIGSIIGOGGDGPGGV) with laminin, fibronectin, fibrinogen, and human serum albumin.

[0178] The storage modulus (G') and loss modulus (G") were measured 30 minutes after uniformly mixing the polypeptide solution with different initiating proteins. As shown in Figure 8, the mechanical properties of the hydrogels initiated by the different proteins were clearly different. In all groups to which the initiating proteins of the present invention were added, hydrogels with a storage modulus of greater than 300 Pa were formed in the mixed systems. This was at least twice the storage modulus of the hydrogels initiated by human serum albumin. This indicates that the self-assembling peptides of the present invention have stronger mechanical strength, i.e., stronger support function, than the responsive hydrogels formed with laminin, fibronectin, and fibrinogen, and the responsive hydrogels formed with the self-assembling peptides and human serum albumin, respectively.

[0179] Experiment 3: Measurement of reorganization properties of the hydrogel of the present invention after deformation Experimental Method: A shear thinning test was carried out to verify the reorganization performance of the hydrogel of the present invention after deformation.

[0180] Preparation of hydrogel samples: 1 wt% of the self-assembling peptide solution was uniformly mixed with 1 wt% of each of laminin, fibronectin, fibrinogen, vitronectin, gamma globulin, transferrin, hemoglobin, and human collagen solutions at a 1:1 volume ratio to obtain a mixed system with a final self-assembling peptide concentration of 1 wt%.

[0181] During testing, 200 μL of the mixture was added to a 20 mm diameter panel of a MARS 60 rheometer and a time scan was performed. Shear thinning was then performed for 20 seconds under conditions of a rotor-to-panel gap of 500 μm, 100% shear strain, and 1 Hz frequency. The shear thinning was then stopped, the shear strain was immediately adjusted to 1%, and a time scan test was immediately performed to monitor the recovery of the hydrogel's mechanical strength. Each 20-second shear thinning test constituted one set of experiments, and each set of experiments was repeated twice to verify the reorganization ability of the hydrogel of the present invention after multiple external stress deformations. The results are shown in Figure 9 for a mixture of the self-assembling peptide represented by SEQ ID NO. 13 (IIIIIGOGIIGPGGEGPGGE) and tissue fluid (containing large amounts of proteins such as laminin, fibronectin, and vitronectin).

[0182] As shown in Figure 9, the hydrogels self-assembled in response to tissue fluid exhibited the property of rapidly re-assembling after being disrupted and deformed by external force, and this property did not disappear with increasing disruption (Figure 9). Upon 100% shear strain, some of the non-covalent bonds within the hydrogel, such as hydrogen bonds, were broken, reducing its mechanical strength and causing it to become liquid. After the external force was removed, the partially disrupted nanofiber clusters rapidly reassembled, rapidly recovering their original mechanical strength. As shown in Figure 9, even after multiple shear thinnings, the hydrogels of the present invention were able to re-self-assemble. The self-assembly time and strength of the re-assembled hydrogels increased with increasing shear thinning cycles, with only a slight loss.

[0183] Because proteins such as laminin, fibronectin, and vitronectin are abundant in tissue fluid, the protein-responsive peptides of the present invention can be rapidly activated to form hydrogels, which exhibit shear thinning and self-repair properties and can be injected. When applied to cell culture, the hydrogels of the present invention can achieve multiple transfers, which is advantageous for cell dispensing and cell fluid exchange, and when applied to the biomedicine field, they can achieve multiple injections.

[0184] Experiments on self-assembling peptides designated by other sequence numbers gave results similar to those of this example, and therefore detailed explanations will be omitted here.

[0185] Example 7 Application of the peptide hydrogel material of the present invention to the biomedicine field Experiment 1: Cell support effect of the hydrogel of the present invention Experimental method: Adherent HepG2 liver cancer cells were cultured in a cell culture flask using high-sugar DMEM medium containing 10% serum in a mild, humid environment at 37°C with 5% CO2. After culturing, the cells were harvested and resuspended in PBS buffer solution at a concentration of 5 × 10 5A cell suspension of 1000 cells / mL was obtained. The prepared cell suspension and Calcein-AM / PI staining solution were mixed uniformly at a volume ratio of 2:1 and incubated at 37°C in the dark for 15 minutes. Pre-prepared hydrogels containing fibrinogen, transferrin, and gamma globulin were added to a glass-bottom culture dish (see Example 3 for preparation). The cell suspension, incubated away from light, was then added and mixed uniformly to bring the total volume in the glass-bottom culture dish to 200 μL, with a final concentration of the self-assembling peptide of 0.1 wt%. When preparing the control sample, the hydrogel material of the present invention in the experimental group was replaced with an equal volume of PBS buffer. Each experimental sample was then observed using an LSM 980 with Airyscan2 high-speed super-resolution laser confocal microscope. The results are shown in Figure 10, using a mixture of the self-assembling peptide represented by SEQ ID NO. 13 (IIIIIGOGIIGPGGEGPGGE) with fibrinogen, transferrin, and gamma globulin, respectively, as an example.

[0186] As can be seen from the results in Figure 10, in the control group that did not contain the hydrogel of the present invention, all of the cells naturally sank to the bottom of the glass-bottom culture dish, were unsupported, and were distributed in a 2D plane. In contrast, in the case of the hydrogel formed by introducing self-assembling peptides into the system and self-assembling in response to fibrinogen, transferrin, and gamma globulin, all of the cells in the system were supported by the three-dimensional network scaffold formed by self-assembly of the self-assembling peptides, and showed a three-dimensional distribution in a 3D environment.

[0187] Experimental Conclusion: This experimental phenomenon proves that the hydrogel material of the present invention, i.e., the three-dimensional network scaffold formed by the self-assembly of self-assembling peptides in response to proteins, can effectively support cells, favoring the realization of in vitro cell growth and culture in a three-dimensional space and preventing cells from sinking, adhering, and growing in a two-dimensional environment. Furthermore, the conditions for the entire initiation process are mild, no additional substances are required, and the use is effective and convenient, without causing damage to cells. Therefore, the polypeptide material of the present invention shows great potential in the fields of cell culture and biomedicine.

[0188] Experiments on self-assembling peptides designated by other sequence numbers gave results similar to those of this example, and therefore detailed explanations will be omitted here.

[0189] Experiment 2: Application of 3D cell preservation Experimental method: This experiment uses storage of mouse mesenchymal stem cells at 4°C as an example. The isolated mouse mesenchymal stem cells were pipetted evenly and dispensed into cryopreservation tubes in 10 ml portions, so that the cell concentration was 1 x 10 6 The cells were pipetted uniformly to a concentration of approximately 1 / mL. For the 3D group, mouse serum and 1% double antibody in a 50% MAP + 50% SFEM solution containing polypeptide SEQ ID NO:20 (FLIVIGOGIIGOGGEGPGGE) were added to a final concentration of 0.05%. For the 2D group, mouse serum and 1% double antibody in a 50% MAP + 50% SFEM solution were added, and then the cells were stored in a refrigerator at 4°C.

[0190] Cell viability test: (1) The preserved cells were mixed uniformly and thoroughly pipetted, and 500 μL of each cell suspension was placed in a 1.5 mL EP tube, washed with PBS, and centrifuged at 1500 rpm for 10 minutes. (2) The supernatant was discarded, and the cells were resuspended in 150 μL of 1% PBA solution. Then, 150 μL of live / dead cell staining working solution was added, mixed thoroughly and homogenously, and incubated at 37°C for 15 minutes in the dark, and detected using a flow cytometer.

[0191] Experimental Results: To determine changes in mesenchymal cell viability during storage, double staining was performed using a Calcein-AM / PI live / dead dual staining kit and detected using a flow cytometer. As shown in Figure 11, from day 3 of storage, the viability 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%). Furthermore, even on day 5 of storage, high cell viability was observed (71% vs. 43%), representing an approximately 30% improvement in viability. This indicates that the storage effect of the mesenchymal stem cell storage system of the present invention is better than that of conventional 2D storage, and cell viability was significantly improved.

[0192] Experiments on self-assembling peptides designated by other sequence numbers gave results similar to those of this example, and therefore detailed explanations will be omitted here.

[0193] Experiment 3: Application of 3D culture of liver cancer cells Adherent cells have the self-reliance to aggregate and form spheres under conditions that inhibit their adhesion to a surface, i.e., they grow in a three-dimensional space and easily become cell spheres. The cell spheres contain concentration gradients of oxygen, nutrients, and metabolic waste, which can mimic the various properties of solid tissues. This makes them an important 3D physiological model for studying solid tumor development and stem cell differentiation, and is widely used in the biomedical field. Furthermore, cell spheres are simpler than other 3D physiological models, allowing imaging analysis using common experimental tools such as optical, fluorescence, and confocal microscopes, simplifying the experimental process. This experiment used adherent HepG2 cells cultured in the self-assembling peptide culture system of the present invention initiated in a high-sugar medium. The formation of cell spheres demonstrated the feasibility of in vitro cell culture that resembles the living organism.

[0194] In experiment 2, the concentration was 1 × 10 6 The method for preparing a cell suspension with a concentration of 1 x 10 cells / mL 5 A cell suspension of 1 × 10 cells / mL was prepared. Using SEQ ID NO: 3 (IIIIIGSIIGOGGEGPGGV) as an example, the resulting concentration was 1 × 10 5A cell suspension of 100 cells / mL was seeded into a 96-well plate containing the self-assembling peptide of the present invention (the final concentrations of the self-assembling peptide in each well were 0.3 wt.%, 0.5 wt.%, and 0.7 wt.%). During the culture period, the upper medium in the well plate was removed and replaced with fresh medium every three days to perform a liquid change.

[0195] After 3, 7, 11, and 15 days of culture, the diameters of 50 cell spheres in the culture system were observed and recorded using an inverted microscope. The diameter data for the cell spheres collected over these three days were averaged and plotted on a graph (Figure 12). To determine whether HepG2 cells cultured with the self-assembling peptide of the present invention could proliferate for long periods in vitro, characteristic cell spheres were photographed (Figure 12). As can be seen from Figure 12, in the culture systems containing 0.3 wt.%, 0.5 wt.%, and 0.7 wt.% initiated self-assembling peptide of the present invention, HepG2 cells were supported and then proliferated in the form of cell spheres. As the culture time increased, the cell spheres became increasingly tight, and their diameters consistently increased at a similar rate, indicating that HepG2 cells could proliferate stably at a uniform rate over a 15-day culture period. This indicates that the self-assembling peptide culture system initiated with a protein-based mixture of the present invention can be used for long-term in vitro three-dimensional cell culture. The relatively uniform growth rate of the cells also indicated good conditions during the culture period and no damage to the cells.

[0196] Experiments on self-assembling peptides designated by other sequence numbers gave results similar to those of this example, and therefore detailed explanations will be omitted here.

[0197] Experiment 4: Application of 3D culture of porcine muscle satellite cells The culture of porcine muscle satellite cells was similar to the 3D culture of liver cancer cells in Experiment 3. In Experiment 2, the concentration was 1 × 10 6 The method for preparing a cell suspension with a concentration of 1 x 10 cells / mL 5A cell suspension of 1 × 10 cells / mL was prepared. Using SEQ ID NO: 21 (IVIVIGOGIIGOGGDGOGGV) as an example, the resulting concentration was 1 × 10 5 A cell suspension of 100 cells / mL was seeded 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.%). During the culture period, the upper medium in the well plate was removed and replaced with fresh medium every 3 days to perform a liquid change.

[0198] After each of the first, second, third, and fourth days of culture, the diameters of 50 cell spheres in the culture system were observed and recorded using an inverted microscope. The diameter data for the cell spheres collected over these three days were calculated as an average and plotted on a graph (Figure 13). To determine whether porcine muscle satellite cells cultured with the self-assembling peptide of the present invention could proliferate for long periods in vitro, characteristic cell spheres were photographed (Figure 13). As can be seen from Figure 13, in the mixed culture system containing 0.3 wt.% of the polypeptide of the present invention and a protein-based substance, porcine muscle satellite cells were supported and then grew and proliferated in the form of cell spheres. As the culture time increased, the cell spheres became increasingly tight, and their diameters consistently increased at a similar rate, indicating that the porcine muscle satellite cells proliferated stably at a uniform rate over the four-day culture period. In other words, the self-assembling peptide of the present invention formed a 3D support structure after being initiated by the mixed culture system containing a protein-based substance, and can be used for long-term in vitro three-dimensional cell culture. The relatively uniform growth rate of the cells indicated good conditions during the culture period and also indicated that the cells were not damaged.

[0199] Experiments 1-4 of this example all demonstrated that the self-assembling peptide culture system initiated by the protein-based material of the present invention has excellent cell support capacity, and Experiment 2 demonstrated that the self-assembling peptide initiated by the protein-based material of the present invention has good biocompatibility and can be used for cell preservation. Experiments 3 and 4 each demonstrated that adding the self-assembling peptide of the present invention to a cell culture medium containing a natural protein-based material can highly reproduce the in vivo growth environment of cells and enable long-term three-dimensional cell culture in vitro.

[0200] Example 8 Application of the hydrogel of the present invention as a dispersible filler for medical use Experimental method: The synthesized self-assembling peptide represented by SEQ ID NO. 25 (IIIIIIGOGIIGOGGEGPGGV) was dissolved in deionized water and vortexed until fully dissolved to obtain a solution with a self-assembling peptide concentration of 1.5%. The solution pH was adjusted to 7.4 with 0.1 M NaOH solution. Figure 14A shows, from left to right, the polypeptide solution, the precipitated poly(L-lactic acid) microsphere solution, and the mixture of the two. The polypeptide solution used was sterilized by high-pressure sterilization, then mixed with L-polylactic acid microspheres (particle size between 25 and 60 μm) and finally dispensed to obtain a self-assembling peptide mixture injection containing 5% microspheres for injection. The solution became opaque, and it was confirmed that the microspheres were uniformly and stably distributed in the self-assembling peptide solution (Figure 14B). After leaving the obtained self-assembling peptide mixture injection containing microspheres at room temperature for one month, all of the obtained injections were in a uniform suspension state, with no obvious solid-liquid separation. After adding 1 / 5 of the tissue fluid, it was confirmed that a hydrogel phenomenon occurred (Figure 14C).

[0201] Similarly, using the same method steps, SEQ ID NO. 27 (FLIVIGSIIGOGAEGPGGV) at a concentration of 1.5% was also successfully supported on 5% polycaprolactone (PCL) microspheres. Figure 15A shows, from left to right, a transparent SEQ ID NO. 27 polypeptide solution, a precipitated polycaprolactone (PCL) microsphere solution, and a mixture of the two. High-temperature sterilized polypeptide solution was mixed with polycaprolactone (PCL) microspheres (particle size between 25 and 50 μm) to achieve a 5% polycaprolactone (PCL) microsphere content. The microspheres were observed to be uniformly and stably distributed in the self-assembling peptide solution (Figure 15B). After allowing the resulting self-assembling peptide mixture to stand at room temperature for one month, all the resulting injections remained in a uniform suspension with no obvious solid-liquid separation. After adding 1 / 5 of the tissue fluid, gelation was observed (Figure 15C).

[0202] In addition to SEQ ID NOs. 25 and 27, the self-assembling peptides shown in SEQ ID NOs. 1-7 and 9-32 of the present invention can all have similar functions for various other cosmetic medical regenerative microspheres (Table 2).

[0203] [Table 2]

[0204] Experimental conclusion: This experimental phenomenon proves that the hydrogel material of the present invention, i.e., a three-dimensional network scaffold self-assembled in response to proteins by protein-responsive self-assembling peptides, can effectively support cosmetic medical microspheres, provide good dispersion and support for the microspheres before initiation, and can rapidly form a hydrogel after being initiated by proteins in tissue fluid, and further exert the function of local molding in tissue. No additional components are required throughout the entire experimental process, making the operation convenient.

[0205] Experiments on self-assembling peptides designated by other sequence numbers gave results similar to those of this example, and therefore detailed explanations will be omitted here.

[0206] The above examples are merely for the purpose of illustrating the technical ideas and features of the present invention, and are intended to enable those skilled in the art to understand and practice the contents of the present invention, and are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit and essence of the present invention shall 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, the hydrophilic domain comprising at least two consecutive β-turn regions capable of forming a β-turn.

2. The protein-responsive self-assembling peptide of claim 1, wherein the at least one β-turn region contains or is linked to one or more acidic amino acids, preferably one acidic amino acid, at a terminal end.

3. The β-turn region contains a β-turn motif formed by 3 to 6 amino acids, and the β-turn motif has the following structure: X 1 X 2 X 3 、X 1 X 2 X 3 X 4 、X 1 X 2 X 3 X 4 X 5 、 or X 1 X 2 X 3 X 4 X 5 X 6 、 Here, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 is an amino acid residue, and in each β-turn motif, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 The protein-responsive self-assembling peptide of claim 1 , wherein each of

4. The protein-responsive self-assembling peptide of claim 3, which comprises one hydroxyproline (O), and preferably said hydroxyproline (O) is contained in said β-turn motif.

5. The protein-responsive self-assembling peptide of claim 1, wherein the hydrophilic domain comprises 2 to 8 β-turn regions.

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

7. The hydrophilic domain has at least one X 2 is hydroxyproline O, or the hydrophilic domain comprises a β-turn motif in which at least one X 2 The protein-responsive self-assembling peptide of claim 3, which comprises a β-turn motif in which is proline P.

8. The X 1 , X 3 and X 4 one or more of X is glycine (G), and / or 3 and X 4 The protein-responsive self-assembling peptide of claim 3 , wherein one or two of

9. The β-turn motif is The present invention relates to a method for producing a polypeptide comprising 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.: 41), GOAA (SEQ ID NO.: 42), GOG, and GOGV (SEQ ID NO.: 43), Preferably, the β-turn motif is 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.: 53) NO.: 54), GOGGK (SEQ ID NO.: 55), GPGAE (SEQ ID NO.: 56), GOGAD (SEQ ID NO.: 56) 2. The protein-responsive self-assembling peptide of claim 1, having an amino acid sequence selected from the group consisting of GPAAD (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), and GOGVI (SEQ ID NO.: 64).

10. The protein-responsive self-assembling peptide of claim 1, wherein the C-terminus of the hydrophilic domain is modified with a reagent or group selected from carboxylic acid, thiol, ketoacid salt, nitrite, phosphonate, sulfite phosphate, carbonate, sulfate, nitrate, vinyl sulfone, amide, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, alkene, ester, thioester, aryl, and / or silane.

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

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

13. The hydrophobic domain is LLLL (SEQ ID NO.: 65), FIII (SEQ ID NO.: 66), III (SEQ ID NO.: 67), III (SEQ ID NO.: 68), ILILI (SEQ ID NO.: 69), FLFLF (SEQ ID NO.: 70), IVIVI (SEQ ID NO.: 71), VIVIV (SEQ ID NO.: 72), VLFIIV (SEQ ID NO.: 73), VLIII (SEQ ID NO.: 74), IVALF (SEQ ID NO.: 74) NO.: 75), LFIVL (SEQ ID NO.: 76), FIAIV (SEQ ID NO.: 77), FIIIV (SEQ ID NO.: 77) 2. The protein-responsive self-assembling peptide of claim 1, having an amino acid sequence selected from Ac-VLFIIV (SEQ ID NO.: 78), Ac-IVIVI (SEQ ID NO.: 80), Ac-IIII (SEQ ID 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 self-assembling peptide further comprises a linking domain; Preferably, the linking domain comprises 2-8, preferably 4-5, amino acid residues; Preferably, the linking domain comprises amino acids with small side chains, amino acids with hydroxyl groups in the side chains, and / or hydrophobic amino acids spaced from the hydrophobic domain; Preferably, the amino acids with small side chains are selected from glycine (G), alanine (A), and serine (S), the amino acids with hydroxyl groups in their side chains are selected from serine (S), threonine (T), and hydroxyproline (O), and the hydrophobic amino acids distant 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; Preferably, the linking domain has an amino acid sequence selected from: 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; More preferably, the linking domain is 2. The protein-responsive self-assembling peptide of claim 1, having an amino acid sequence selected from 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.: 94), OGII (SEQ ID NO.: 96), GOGVI (SEQ ID NO.: 102), or GOGII (SEQ ID NO.: 103).

15. 2. The protein-responsive self-assembling peptide of claim 1, wherein the protein-responsive self-assembling peptide has an amino acid sequence selected from the following SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32.

16. 16. A method for forming a scaffold material from the protein-responsive self-assembling peptide of any one of claims 1 to 15, the method comprising initiating the self-assembling peptide with a protein-based substance or a mixed system comprising a protein-based substance to form a scaffold material; Preferably, the method includes a step of mixing the self-assembling peptide with a protein-based substance or a mixed system containing a protein-based substance, or the method includes a step of injecting or implanting the self-assembling peptide into a mixed system containing the protein-based substance.

17. The protein-based 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 of less 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 protein-based substance is selected from laminin, fibronectin, fibrinogen, globulin, hemoglobin, vitronectin, transferrin, or a combination comprising one or more of these; The method according to claim 16, wherein the mixed system containing the protein-based substance is selected from serum, plasma, cell culture medium, animal or plant tissue fluid, or animal tissue.

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

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

21. A composition comprising the protein-responsive self-assembling peptide of any one of claims 1 to 15 and a protein-based substance or a mixed system containing a protein-based substance of claim 16.

22. The protein-responsive self-assembling peptide according to any one of claims 1 to 15, the method according to any one of claims 16 to 18, the scaffold material according to claim 19 or 20, or the composition according to claim 21, is used in one or more applications selected from the group consisting of regenerative medicine and tissue regeneration, 2D and 3D cell culture and preservation, microsphere dispersion and embedding, drug delivery, wound healing, implant materials, gene therapy, stem cell therapy, and aesthetic medicine.

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