Self-assembling peptide and application thereof

Abstract

The invention relates to a self-assembling peptide, a three-dimensional network material formed by the self-assembling peptide and application of the three-dimensional network material. The self-assembling peptide of the present invention comprises a hydrophilic domain comprising at least one beta-turn region capable of forming a beta-turn, and an RGD domain comprising an amino acid sequence of arginine (R)-glycine (G)-aspartic acid (D). The self-assembled peptide can be self-assembled to form a three-dimensional network material in a neutral solution without being started by any other exogenous components.

Description

Technical Field

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

[0002] Self-assembling peptides have attracted much attention due to their good biocompatibility and diverse functions. Peptides are composed of amino acids through amide bonds. Since amino acid residues have different chemical structures, peptides can effectively realize molecular self-assembly by utilizing hydrogen bonding between peptide bonds and hydrogen bonding between amino acid residues, electrostatic effects, hydrophobic effects, and π-π stacking effects, thereby spontaneously forming aggregates with certain structures under specific responses.

[0003] The network structure formed by peptide self-assembly can highly reproduce the natural extracellular matrix (ECM) niche, providing synergistic biochemical and biophysical cues to guide cell proliferation, migration and differentiation. It also has the characteristics of high water content, biocompatibility, shear thinning, rapid self-repair after damage, and is injectable. It is an ideal system for cell culture and storage, organoid cultivation and implantation, tissue repair, etc., and has attracted more and more attention in stem cells, tissue engineering and even the entire biomedicine field.

[0004] Most polypeptide self-assembled network materials are in a hydrogel state when used, which is not convenient for cell inoculation and separation; the formation of most network structures depends on high concentrations of salt ions, acidic pH environment, certain specific proteins, light, or temperatures that are not suitable for cell survival, which is not conducive to application scenarios such as cell storage, culture or human implantation.

[0005] Therefore, it is of great significance to develop self-assembling peptides that can self-assemble into nano-network structures under physiological conditions, cell storage and culture environments, and substances widely present in the human body, and that can provide support and repair functions to cells, functional microspheres, functional molecules, and drugs in both solution and hydrogel states, and to develop their applications in the fields of 3D cell culture, medical aesthetic regeneration and repair, and implant carriers. Summary of the invention

[0006] The invention provides a self-assembling peptide containing an RGD cell adhesion sequence, which can self-assemble to form a three-dimensional network material in a neutral solution without any other exogenous components.

[0007] In a first aspect, the present invention provides a self-assembling peptide, which comprises a hydrophilic domain and an RGD domain, wherein the hydrophilic domain comprises at least one β-turn region capable of forming a β-turn, and the RGD domain comprises an amino acid sequence of arginine (R)-glycine (G)-aspartic acid (D).

[0008] In some embodiments, the at least one β-turn region comprises or is linked to one or more acidic amino acids or hydrophobic amino acids at the terminal end, preferably comprises or is linked to one acidic amino acid or hydrophobic amino acid.

[0009] In some embodiments, at least one β-turn region comprises an acidic amino acid or a hydrophobic amino acid at a terminus.

[0010] The acidic amino acids of the present invention include but are not limited to aspartic acid (D) or glutamic acid (E), and the hydrophobic amino acids include but are not limited to alanine (A), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), and valine (V).

[0011] 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:

[0012] X1X2X3, X1X2X3X4, X1X2X3X4X5, or X1X2X3X4X5X6,

[0013] Among them, X1, X2, X3, X4, X5, and X6 are amino acid residues, and X1, X2, X3, X4, X5, and X6 in each β-turn motif are the same as or different from each other.

[0014] In some embodiments, the self-assembling peptide comprises one hydroxyproline (O).

[0015] In some embodiments, the β-turn motif comprises one hydroxyproline (O), and preferably, X2 is hydroxyproline (O).

[0016] In some embodiments, the hydrophilic domain comprises 1-8 β-turn regions.

[0017] In some embodiments, the hydrophilic domain comprises 1, 2, 3, 4, 5, 6, 7, or 8 β-turn regions.

[0018] Preferably, the hydrophilic domain comprises 1-6 β-turn regions. More preferably, the hydrophilic domain comprises 1-4 β-turn regions. Preferably, the hydrophilic domain comprises 1, 2 or 3 β-turn regions.

[0019] In some embodiments, the β-turn motif in the β-turn region comprises or is linked to an acidic amino acid.

[0020] In some embodiments, the β-turn motif in the β-turn region comprises or is linked to a glutamic acid (E), valine (V), leucine (L), isoleucine (I), aspartic acid (D) or lysine (K).

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

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

[0023] In some embodiments, the hydrophilic domain comprises two β-turn regions, and the ends of the β-turn regions comprise an acidic amino acid D.

[0024] In some embodiments, the hydrophilic domain comprises two β-turn regions, wherein one β-turn region comprises an acidic amino acid D at one end and the other β-turn region comprises an amino acid V at one end.

[0025] In some embodiments, the hydrophilic domain comprises a β-turn region, and the end of the β-turn region comprises an acidic amino acid E.

[0026] In some embodiments, the hydrophilic domain comprises two β-turn regions, wherein one β-turn region comprises an acidic amino acid E at one end and the other β-turn region comprises an amino acid V at one end.

[0027] In some embodiments, the hydrophilic domain comprises a β-turn region, and the end of the β-turn region comprises an acidic amino acid D.

[0028] In some embodiments, the hydrophilic domain comprises a β-turn region, wherein a terminus of the β-turn region comprises amino acid V.

[0029] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X2 is hydroxyproline O, or the hydrophilic domain comprises at least one β-turn motif in which X2 is proline P.

[0030] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X2 is O.

[0031] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X1 is G.

[0032] In some embodiments, the hydrophilic domain comprises a β-turn motif in which X2 is O, and a β-turn motif in which X2 is P.

[0033] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X2 is P.

[0034] In some embodiments, the hydrophilic domain comprises two β-turn motifs in which X2 is P.

[0035] In some embodiments, one or more of X1, X3 and X4 is glycine (G).

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

[0037] GPGGE (SEQ ID NO:66), GOGAE (SEQ ID NO:67), GOGGAE (SEQ ID NO:68),

[0038] GOGE(SEQ ID NO:69), GOGGE(SEQ ID NO:70), GPGAD(SEQ ID NO:71),

[0039] GOGGD (SEQ ID NO:72), GPGGV (SEQ ID NO:73), GOGGV (SEQ ID NO:74),

[0040] GPGGK (SEQ ID NO:75), GOGGK (SEQ ID NO:76), GPGAE (SEQ ID NO:77),

[0041] GOGAD (SEQ ID NO:78), GPAAD (SEQ ID NO:79), GOAAE (SEQ ID NO:80),

[0042] GPGGD (SEQ ID NO:81), GPGGGV (SEQ ID NO:82), GPGV (SEQ ID NO:83),

[0043] OGGAE (SEQ ID NO: 84) or GOGVI (SEQ ID NO: 88).

[0044] In some embodiments, X1 and X4 form a hydrogen bond.

[0045] In some embodiments, the hydrophilic domain comprises 1, 2, 3, 4, 5, 6, 7 or 8 β-turn motifs, preferably, the hydrophilic domain comprises 1 or 2 or 3 β-turn motifs.

[0046] At least one beta turn motif in the hydrophilic domain includes an alanine, thereby improving the mechanical properties of the self-assembling peptide.

[0047] In some embodiments, the N-terminus of the hydrophilic domain can be modified with an agent or group selected from the following: carboxylic acid, thiol, ketoate, nitrite, phosphonate, phosphite, carbonate, sulfate, nitrate, vinyl sulfone, amide, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and / or silane modification.

[0048] In some embodiments, the RGD domain comprises or is linked to one or more amino acids selected from glycine (G), proline (P), or hydroxyproline (O).

[0049] In some embodiments, the RGD domain comprises or is linked to an amino acid selected from glycine (G)-proline (P). In some embodiments, the RGD domain comprises or is linked to an amino acid selected from glycine (G)-hydroxyproline (O).

[0050] In some embodiments, the self-assembling peptide further comprises a hydrophobic domain. The amino acid sequence of the hydrophilic domain is more hydrophilic than the amino acid sequence of the hydrophobic domain.

[0051] In some embodiments, the hydrophobic domain comprises 3-10 hydrophobic amino acids. Preferably, the hydrophobic domain comprises 3-7 hydrophobic amino acids, preferably, the hydrophobic domain comprises 3-5 hydrophobic amino acids. More preferably, the hydrophobic domain comprises 5 hydrophobic amino acids.

[0052] Preferably, the hydrophobic amino acid is selected from one or more of isoleucine (I), valine (V), leucine (L), phenylalanine (F) and alanine (A).

[0053] In some embodiments, the hydrophobic amino acid is selected from one or more of I, V, L, and F.

[0054] In some embodiments, the C-terminus of the hydrophobic domain is modified with a reagent or group selected from the following: acetyl, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and / or silane modification.

[0055] In some embodiments, the hydrophobic domain comprises an amino acid sequence selected from the group consisting of:

[0056] FIIII (SEQ ID NO: 32), IIII (SEQ ID NO: 33), IIII (SEQ ID NO: 34), ILILI (SEQ ID NO: 35),

[0057] FLFLF (SEQ ID NO:36), IVIVI (SEQ ID NO:37), VLFIIV (SEQ ID NO:38),

[0058] VLIII (SEQ ID NO:39), IVALF (SEQ ID NO:40), LFIVL (SEQ ID NO:41),

[0059] FIAIV (SEQ ID NO:42), FIIIV (SEQ ID NO:43), Ac-VLFIIV (SEQ ID NO:44),

[0060] Ac-IVIVI (SEQ ID NO:45), Ac-IIIIII (SEQ ID NO:46), IIIIII (SEQ ID NO:47),

[0061] FLIVI(SEQ ID NO:48), FLIIA(SEQ ID NO:49), FIFIF(SEQ ID NO:50),

[0062] IFIFI (SEQ ID NO:51), IAILI (SEQ ID NO:52), LLLLL (SEQ ID NO:53) or VIVIV (SEQ ID NO:54).

[0063] In some embodiments, the RGD domain is located at the end of the self-assembling peptide, preferably at the end of the self-assembling peptide close to the hydrophilic domain.

[0064] In some embodiments, the RGD domain is located between the hydrophobic domain and the hydrophilic domain.

[0065] The amino acid sequence of the hydrophobic domain is hydrophobic compared to the amino acid sequence of the hydrophilic domain.

[0066] In some embodiments, the self-assembling peptide further comprises a connecting domain, providing a spacer between the hydrophobic domain and the hydrophilic domain, or providing a spacer between the hydrophobic domain and the RGD domain, or providing a spacer between the hydrophilic domain and the RGD domain.

[0067] In some embodiments, the connecting domain comprises 2-8 amino acid residues, preferably 4-5 amino acid residues.

[0068] In some embodiments, the linker domain comprises small side chain amino acids, amino acids with hydroxyl groups on the side chains, and / or hydrophobic amino acids that are far away from the hydrophobic region.

[0069] In some embodiments, the amino acid with a smaller side chain is selected from glycine (G), alanine (A), and serine (S).

[0070] In some embodiments, the amino acid with a hydroxyl group on the side chain is selected from serine (S), threonine (T) and hydroxyproline (O),

[0071] In some embodiments, the hydrophobic amino acids away from the hydrophobic domain are selected from I, V, L, F and A, and the hydrophobic amino acids I, V, F, L, A are interchangeable.

[0072] In some embodiments, the connecting domain comprises an amino acid sequence selected from the group consisting of GSII, GSGII, GSVI, GOII, OGII, or GTVI, wherein S, T, and O are interchangeable with each other.

[0073] More preferably, the connecting domain comprises an amino acid sequence selected from the group consisting of:

[0074] GSII (SEQ ID NO:55), GTII (SEQ ID NO:56), GTVI (SEQ ID NO:57), GOVI (SEQ ID NO:58), GSVI (SEQ ID NO:59), GSGII (SEQ ID NO:60), GSGVI (SEQ ID NO:61), GOII (SEQ ID NO:62), OGII (SEQ ID NO:63), GOGVI (SEQ ID NO:63) ID NO:64) or GOGII (SEQ ID NO:65).

[0075] In some embodiments, one or more Gs are further included between the hydrophobic domain and the connecting domain to enhance the softness and flexibility of the self-assembling peptide.

[0076] In some embodiments, the connecting domain is located between the hydrophobic domain and the hydrophilic domain, or the connecting domain is located between the hydrophobic domain and the RGD domain, or the connecting domain is located between the hydrophilic domain and the RGD domain.

[0077] In some embodiments, the self-assembling peptide has a length of 15-50 amino acids, preferably 15-25 amino acids.

[0078] 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.

[0079] In some embodiments, the self-assembling peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and SEQ ID NOs: 6-29:

[0080] IIIIIIGSIIGPGGDGPRGD (SEQ ID NO:1),

[0081] IIIIIGSIIGPGGEGPRGD (SEQ ID NO:2),

[0082] IIIIIGSIIGOGGEGPRGD (SEQ ID NO:3),

[0083] IIIIGSIIGOGGEGPRGD (SEQ ID NO:4),

[0084] IIIIIGSIIGOGGEGPRGD (SEQ ID NO:6),

[0085] IIIIIIGSIIGORGDGPGGE (SEQ ID NO:7),

[0086] IIIIIIGSIIGOGAEGPRGD (SEQ ID NO:8),

[0087] IIIIIIGSIIGORGDGPGAE (SEQ ID NO:9),

[0088] IIIIIIGSIIGOGGVGPRGD (SEQ ID NO:10),

[0089] IIIIIIGSIIGOGAEGPRGDGGPGGV (SEQID NO:11),

[0090] FLIVIGSIIGOGAEGPRGD (SEQ ID NO:12),

[0091] FLIIAGSIIGPGGDGORGD (SEQ ID NO:13),

[0092] IIIIIIGOGIIGPRGDGPGGE (SEQ ID NO:14),

[0093] FIFIFGTVIGPRGDGOGGE (SEQ ID NO:15),

[0094] IFIFIGTVIGPGGEGORGD (SEQ ID NO:16),

[0095] IAILIGTVIGPRGDGOGGE (SEQ ID NO:17),

[0096] IVIVIGSIIGPRGDGPGGV (SEQ ID NO:18),

[0097] IVIVIGSIIGOGGDGPRGD (SEQ ID NO:19),

[0098] IVIVIGSIIGPGGEGORGD (SEQ ID NO:20),

[0099] FLIVIGOGVIGOGGEGPRGD (SEQ ID NO:21)

[0100] IVIVIGOGVIGOGGVGPRGD (SEQ ID NO:22),

[0101] IVIVIGSGIIGPGGEGPRGD (SEQ ID NO:23),

[0102] IVIVIGOGVIGPRGDGOGGV (SEQ ID NO:24),

[0103] FIIIVGSIIGPGGEGPRGD (SEQ ID NO:25),

[0104] IIIIIIGOGIIGOGGEGPRGD (SEQ ID NO:26),

[0105] Ac-IIIIGSIIGPGGEGORGD (SEQ ID NO:27),

[0106] FLIVIGSIIGOGAEGPRGD (SEQ ID NO:28),

[0107] FLIVIGSIIGOGAEGORGD (SEQ ID NO:29).

[0108] The self-assembling peptide of the present invention having the above structure can be triggered to form a three-dimensional network material.

[0109] The three-dimensional network material has a nanostructure.

[0110] In a second aspect, the present invention provides a method for preparing the self-assembling peptide of the first aspect, which is prepared by a method selected from solid phase peptide synthesis, microbial synthesis, fermentation or other biochemical methods.

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

[0112] Therefore, in a third aspect, the present invention provides a method for forming a three-dimensional network structure by inducing the self-assembling peptide of the first aspect, the method comprising the step of inducing the self-assembling peptide to form a three-dimensional network material under a condition of pH less than 9.

[0113] In some embodiments, the method includes initiating the self-assembling peptide solution under conditions of pH less than 9. In the present invention, the presence of the RGD domain in the self-assembling peptide makes the self-assembly of the self-assembling peptide of the present invention have pH-dependent characteristics. Under different pH initiation conditions, the degree of self-assembly varies with the concentration. When the degree of self-assembly is low, a viscous material is formed, and when the degree of self-assembly is high, a hydrogel material is formed. The self-assembling peptide containing RGD of the present invention can self-assemble itself without having an obvious triggered assembly response to metal ions and proteins. For example, it can self-assemble in response to calcium ions, but not significantly respond to magnesium ions and BSA.

[0114] In some embodiments, the self-assembling peptide solution is initiated at a pH of 6 to 9 or less, for example, at a pH of 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.2, 8.4, 8.6, 8.8, 8.9 or any value therebetween. In some preferred embodiments, the self-assembling peptide solution is initiated at a pH of 6-7.5.

[0115] In some embodiments, the solvent that triggers self-assembly is a solvent that can adjust the pH value of the self-assembling peptide solution to less than 9, including but not limited to acidic solutions (dilute hydrochloric acid, acetic acid, etc.).

[0116] In some embodiments, in the self-assembling peptide solution, the concentration of the self-assembling peptide is ≤0.09wt%, preferably 0.0001wt%-0.09wt%, more preferably 0.01wt%-0.09wt%, and the initiation is performed to obtain a three-dimensional network material in a solution state.

[0117] In some embodiments, in the self-assembling peptide solution, the concentration of the self-assembling peptide is ≥0.1wt%, preferably >0.3wt%, further preferably 0.3wt%-4wt%, and further preferably 0.5wt%-1wt%, and the initiation is performed to obtain a three-dimensional network material in a hydrogel state.

[0118] In some embodiments, the self-assembling peptide solution can form a hydrogel film layer.

[0119] In some embodiments, the preparation method further comprises: completely dissolving the self-assembling peptide under alkaline conditions, preferably under conditions with a pH value greater than 7.4, more preferably in an aqueous solvent with a pH value greater than 9, to obtain a self-assembling peptide solution.

[0120] The self-assembling peptide can be dissolved in an alkaline solvent, and the pH of the solution can be adjusted after dissolution. The solvent includes one or more aqueous solutions of sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonia water, etc. that can provide an alkaline environment.

[0121] The solvent can be used to dissolve the self-assembling peptides of the present invention as long as it can provide an alkaline environment solution, preferably a physiologically acceptable solution.

[0122] In some embodiments, the preparation method includes: completely dissolving the self-assembling peptide under alkaline conditions (pH greater than 9) to obtain a self-assembling peptide solution, adjusting the pH value of the self-assembling peptide solution to less than 9 (preferably 6-7.5) with an acidic solution (dilute hydrochloric acid, acetic acid, etc.) to obtain the three-dimensional network material; optionally, when the concentration of the self-assembling peptide in the self-assembling peptide solution is ≤0.09wt% (preferably 0.0001wt%-0.09wt%, more preferably 0.01wt%-0.09wt%), the obtained three-dimensional network material is in a solution state; when the concentration of the self-assembling peptide in the self-assembling peptide solution is greater than 0.3wt%, the obtained three-dimensional network material is in a hydrogel state.

[0123] The mechanical strength of the formed hydrogel gradually increases with time, and components such as serum, plasma, and tissue fluid promote and enhance its gelation properties.

[0124] In the hydrogel, the concentration of the self-assembling peptide is ≥ 0.1 wt %, preferably 0.3-4 wt %, and more preferably 0.5-1 wt %.

[0125] The present invention unexpectedly found that the mesh scaffold formed by the self-assembly of the self-assembly peptide can be deposited to form a hydrogel film layer, and the formed three-dimensional mesh scaffold structure is more stable. Therefore, such self-assembly peptides are particularly suitable for the application scenario of culturing cells on the gel after hydrogel coating in 3D cell culture. The three-dimensional network material is a nanostructure.

[0126] At the same time, the hydrogel formed by the self-assembling peptide of the present invention at high concentration has a self-repairing function. The gel state of the hydrogel can be destroyed under the action of mechanical force, and then restored to the gel state after the mechanical force disappears, and the recovery time does not exceed 10 minutes. After recovery, the storage modulus of the hydrogel is at least 70% of that before being destroyed, preferably at least 85% of that before being destroyed, and more preferably more than 95% of that before being destroyed.

[0127] In addition, the hydrogel of the present invention contains a nanofiber three-dimensional network structure formed by self-assembly of a self-assembling peptide or its derivative solution, which can be applied to a variety of application scenarios: in vitro three-dimensional culture, establishment of cell models; loading cells or organoids or organs, and injecting them into animals for tissue repair and other research; as wound dressings, hemostatic materials, etc.; as a dispersion of microspheres containing polylactic acid, polystyrene, etc. and their application in cell 3D culture; or as a carrier for sustained release of drugs or functional factors; or as a cell preservation material; and applied in biotherapy, tissue engineering, and regenerative medicine.

[0128] It should be noted that if the hydrophobicity of the hydrophobic domain is too strong, it is easy to form an association, and if the hydrophobicity of the hydrophobic domain is too weak, self-assembly cannot be achieved. The hydrophilic domain interacts with the initiator to trigger the self-assembly of the self-assembling peptide aqueous solution into a peptide hydrogel. Therefore, the selection of hydrophobic amino acids in the hydrophobic domain, the selection of hydrophilic amino acids in the hydrophilic domain, and the balance and precise matching of hydrophobic amino acids and hydrophilic amino acids are particularly important for the self-assembling peptide of the present invention not to form a hydrogel under neutral conditions and to form a hydrogel under initiation conditions. The RGD domain contains a tripeptide sequence of arginine-glycine-aspartic acid, which can enhance the adhesion of cells in application scenarios such as cell culture and stem cell implantation, while functionally simulating the real extracellular matrix in the cell 3D culture chamber.

[0129] The present invention unexpectedly found that the self-assembling peptide having the structure of the present invention can be triggered and self-assembled to form a nano-network structure under physiological conditions, cell storage and culture environments, and the presence of a wide range of endogenous substances in the human body. In particular, using endogenous physiological substances, such as serum, plasma, cell culture medium, animal and plant tissue fluid, and animal tissue as initiators greatly reduces the safety risks in clinical applications, which is particularly important for drug delivery, human tissue repair, and regenerative medicine.

[0130] In a fourth aspect, the present invention provides a three-dimensional network material, wherein the three-dimensional network material comprises the self-assembling peptide of the first aspect.

[0131] In some embodiments, the three-dimensional network material is obtained using the method of the third aspect.

[0132] In some embodiments, the three-dimensional network material is in a solution state. Preferably, in the three-dimensional network material, the concentration of the self-assembling peptide is ≤ 0.1 wt %; more preferably 0.0001 wt %-0.09 wt %; further preferably 0.01 wt %-0.09 wt %.

[0133] In some embodiments, the three-dimensional network material is in a hydrogel state. Preferably, in the three-dimensional network material, the concentration of the self-assembling peptide is ≥ 0.1 wt%, preferably > 0.3 wt%, and more preferably 0.3 wt%-4 wt%.

[0134] In some embodiments, the three-dimensional network material is in a dry powder state. Preferably, the three-dimensional network material in a hydrogel state is obtained by freeze drying, spray drying, or drying.

[0135] In some embodiments, the three-dimensional network material is in the form of a hydrogel or in a dried form of a hydrogel, such as in the form of a lyophilized powder of a hydrogel.

[0136] In some embodiments, the three-dimensional network material is in the form of an injectable hydrogel.

[0137] In some embodiments, the three-dimensional network material is a nanostructure.

[0138] The three-dimensional network material has more β-folded structures than the self-assembling peptide.

[0139] In a fifth aspect, the present invention provides a composite system comprising the self-assembling peptide of the first aspect and a microcarrier.

[0140] In some embodiments, the microcarrier is a microsphere or a microslide.

[0141] In some embodiments, the microcarrier includes at least one of polystyrene microcarriers, polylactic acid microcarriers, macroporous gelatin microcarriers, methacrylate gelatin microcarriers, laminarin microcarriers, carboxybetaine microcarriers, isopropylacrylamide microcarriers, polyvinyl alcohol / cellulose microcarriers, D,L-lactic acid-co-ethylene glycol microcarriers, D,L-lactic acid-co-ethylene glycol microcarriers, D,L-lactic acid-co-ethylene glycol microcarriers, D,L-lactic acid-co-ethylene glycol / chitosan microcarriers, collagen microcarriers, chitosan microcarriers, chitin microcarriers, PHEMA microcarriers, polyurethane foam microcarriers, polylactide microcarriers, alginate gel microcarriers and magnetic microcarriers.

[0142] In some embodiments, the particle size of the microcarrier is 50-500 μm, preferably 150-500 μm.

[0143] In some embodiments, the self-assembling peptide and the microcarrier are placed in the same container in the form of powder; or, the self-assembling peptide and the microcarrier are placed in different containers in the form of powder; or, the self-assembling peptide is placed in one container in the form of a solution, and the microcarrier is placed in another container in the form of a powder; or, the self-assembling peptide is placed in one container in the form of a powder, and the microcarrier is placed in another container in the form of a solution; or the self-assembling peptide and the microcarrier are placed in the same container in the form of a solution.

[0144] In a sixth aspect, the present invention provides a cell culture system, comprising the microcarriers in the composite system of the fifth aspect and a three-dimensional network material formed by self-assembly of self-assembling peptides, wherein the microcarriers are supported by the three-dimensional network material.

[0145] In some embodiments, the concentration of the microcarriers is 0.05-10.0 wt %, the concentration of the three-dimensional network material is 0.025-1.0 wt %, and the mass ratio of the three-dimensional network material to the microcarriers is 1-20:1-20.

[0146] In a seventh aspect, the present invention further provides a method for culturing cells, the method comprising the step of culturing cells using the composite system of the fourth aspect or the cell culture system of the fifth aspect.

[0147] Before cell culture, the self-assembling peptide is prepared into a solution and then added into the microcarrier cell culture system, so that the three-dimensional network material formed by the self-assembling peptide can support the microcarrier for cell culture.

[0148] In some embodiments, the cell culture system is in the form of a solution, and the corresponding minimum three-dimensional network material concentration can be selected for different microcarrier content concentrations. A higher three-dimensional network material concentration can support more microcarriers.

[0149] In the cell culture method of the present invention, cells are attached to microcarriers, and the microcarriers are supported by three-dimensional network materials formed by self-assembly of self-assembling peptides in the system. Although it is a static culture, it is similar to dynamic culture, and the microcarriers are suspended in the system, evenly distributed and remain static.

[0150] The cell culture system of the present invention can provide a novel microcarrier culture cell mode, that is, the microcarrier is supported by a three-dimensional network material, and the uniform dispersion and suspension state of the microcarrier are achieved, so as to better help cell adhesion and promote cell proliferation, and the microcarrier is always kept in a state of uniform suspension and dispersion throughout the cell culture cycle, without aggregation or precipitation. This mode combines the advantages of static culture and dynamic culture, integrates these two modes of static culture and dynamic culture into a new mode, and successfully solves some problems of microcarrier culture cells. The present invention conducts cell culture experiments with this new mode and three different experimental schemes of static culture and dynamic culture, and compares them, thereby verifying the feasibility and advantages of this new mode. In this mode, cells adhere to microcarriers, and microcarriers are supported by three-dimensional network materials in the system. Similar to dynamic culture, microcarriers are suspended in the system, without aggregation, precipitation, uniform distribution and keeping static state.

[0151] In some embodiments, the cell is a mammalian cell.

[0152] In some embodiments, the cells include but are not limited to Vero cells, CHO cells, stem cells, BHK cells, SP2 / 0 cells, MDCK, PK15, Marc-145, HEK293 cells, ST cells, cancer cells, and porcine muscle satellite cells.

[0153] In some embodiments, the stem cells are embryonic stem cells, umbilical cord blood stem cells, mesenchymal stem cells, or induced pluripotent stem cells (iPS), and the mesenchymal stem cells are, for example, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, skin mesenchymal stem cells, etc.

[0154] In an eighth aspect, the present invention provides a composition comprising the self-assembling peptide of the first aspect, the composite system of the fifth aspect, and the aqueous solvent and / or acid solution for adjusting pH of the third aspect.

[0155] The composition is in the form of being combined together, or in the form of being combined, the latter being that the self-assembling peptide and other components are placed in different containers respectively.

[0156] In some embodiments, the solvent for dissolving the self-assembling peptide is an alkaline solvent, preferably an alkaline solvent that provides an alkaline environment, preferably an alkaline environment with a pH value greater than 9, including but not limited to: aqueous solutions of one or more of sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonia, etc.

[0157] In some embodiments, the solvent that triggers self-assembly is a solvent that can adjust the pH value of the self-assembling peptide solution to less than 9, preferably an acidic solution, including but not limited to dilute hydrochloric acid, acetic acid, and the like.

[0158] In the ninth aspect, the present party provides the self-assembling peptide of the first aspect, the method of the second aspect, the preparation method of the third aspect, the three-dimensional network material of the fourth aspect, the composite system of the fifth aspect, the cell culture system of the sixth aspect, the method of culturing cells of the seventh aspect, or the composition of the eighth aspect for use in one or more selected from the following: in vitro three-dimensional storage and culture of cells, cell migration test, organoid construction, in vitro three-dimensional storage and culture of viruses, tissue engineering scaffolds or tissue filling materials, microsphere and microcarrier dispersions or embedding fillings, drug, vaccine and compound storage and application carriers, medical dressings or medical cosmetic materials, tissue engineering scaffolds, stem cell transplantation, tumor animal in vivo transplantation, gene therapy, stem cell therapy, tissue repair, medical cosmetology, regenerative medicine and tissue regeneration.

[0159] The three-dimensional network material of the present invention is safe and convenient during preparation, and does not require the adjustment of the temperature, light, salt or ion components of the system. The self-assembly can be further promoted and enhanced by being dissolved in a neutral solution system including a complete cell culture medium, a serum-free culture medium, and a mixed system such as human tissue fluid, thereby better ensuring the biocompatibility of the hydrogel of the present invention. The self-assembly peptide sequence contains an RGD sequence, which can enhance the adhesion of cells in application scenarios such as cell culture and stem cell implantation, while the cell 3D culture chamber functionally simulates the real extracellular matrix.

[0160] The responsive self-assembling peptide provided by the present invention and the three-dimensional network system prepared therefrom can be used for in vitro three-dimensional culture and storage, establishing cell models, loading cells or organs or organoids, and can be injected into animals or humans for tissue repair; can also be used as wound dressings, hemostatic materials, etc.; or as carriers for sustained-release drugs or functional factors; or as cell preservation materials and other biological treatments, tissue engineering, regenerative medicine. In summary, the self-assembling peptide of the present invention and the three-dimensional network system prepared therefrom have a wide range of applications and are safe and convenient. DETAILED DESCRIPTION

[0161] The present invention is further described below in conjunction with specific embodiments. These embodiments are provided by way of illustration only and do not limit the protection scope of the present invention.

[0162] The present invention provides a self-assembling peptide, and also provides a hydrogel obtained by self-assembling the self-assembling peptide under neutral pH conditions, wherein the scaffold material in the form of a hydrogel is a hydrogel material having a three-dimensional network-like scaffold structure. The self-assembling peptide of the present invention can self-assemble in a neutral liquid state, does not require exogenous components, and avoids the risks of introducing other exogenous substances, such as certain metal salt ions, specific proteins, etc.

[0163] The advantages of the present invention are also reflected in that the self-assembling peptide of the present invention can be formulated into a self-assembling peptide solution under neutral conditions. At low concentrations, the cells are still formed into a 3D stereoscopic distribution due to the strong cell support function, and the viscosity is low, thereby maintaining the convenience of operation using the self-assembling peptide solution. The isoelectric point of the self-assembling peptide of the present invention is in the neutral pH range, so the peptide as a whole tends to be uncharged in a neutral solution, thereby reducing the repulsion between the self-assembling peptide molecules. The self-assembling peptide molecules then realize the self-assembly of the self-assembling peptide molecules through hydrophobic interaction and hydrogen bonding, and finally form a nano three-dimensional network structure, and can form a hydrogel at a high concentration (1%). The reason is that in acidic (peptide molecules are positively charged) or alkaline conditions (peptide molecules are negatively charged), there is a charge repulsion between the self-assembling peptide molecules, and a tight molecular stack cannot be formed. Even if the hydrophobic effect of the hydrophobic domain provides power for the aggregation of the self-assembling peptide, the electrostatic repulsion between the molecules hinders the stable and orderly arrangement. In addition, aspartic acid (D) in the hydrophilic domain is a hydrophilic amino acid, which tends to be exposed in the solution. The β-turn structure reduces the interference of other side chains near aspartic acid. Therefore, the β-turn structure is conducive to the role of acidic amino acids. The β-turn structure makes the side chain groups of aspartic acid on the self-assembling peptide more active, aggravates the repulsion between molecules, affects the arrangement of molecules, and further increases the difficulty of the self-assembling peptide to form a three-dimensional mesh scaffold structure. Moreover, when the self-assembling peptide molecules approach each other during the movement, they are given acceleration due to electrostatic effects, which increases the kinetic energy of the entire system. Therefore, in a solution containing only self-assembling peptides under neutral conditions, the distribution of self-assembling peptide molecules is relatively chaotic and cannot form a three-dimensional network material. Once the solution in which such a self-assembling peptide is located is close to its isoelectric point pH, most of the self-assembling peptide molecules are in a state where the overall positive and negative charges are equivalent, the electrostatic repulsion between molecules is reduced, and the hydrophobic effect and hydrogen bonds cause the molecules to aggregate and distribute regularly in an orderly manner to form a three-dimensional network material.

[0164] The concentration of the self-assembling peptide in the hydrogel of the present invention is preferably above 0.1wt%, preferably in the range of 0.1-4wt%, and more preferably in the range of 0.1-1wt%. The self-assembling peptide of the present invention responds quickly to pH. When the concentration of the self-assembling peptide is greater than 1.5%, within a short period of time, preferably within 30 minutes, preferably within 15 minutes, or even within 10 minutes after the self-assembling peptide solution is adjusted to a neutral pH, the two self-assemble into a hydrogel through non-covalent cross-linking, that is, gelation is achieved.

[0165] The self-assembling peptide of the present invention not only has the advantage of rapidly assembling to form a hydrogel in a short time, but also has the characteristics of shear thinning and rapid recovery to the original state after removing the external force. This shows that under the action of strong external mechanical force, some non-covalent bonds inside the hydrogel will break, and the macroscopic manifestation is that the gel state is destroyed and becomes a solution; when this mechanical force disappears, these broken non-covalent bonds will reconnect, and the macroscopic manifestation is that the destroyed gel recovers to a state similar to the original gel through self-repair. And the recovery time of the hydrogel does not exceed 10 minutes, and the storage modulus after recovery is at least 70% of the original, and can even reach more than 85% of the original, or even more than 95%. Further, the hydrogel of the present invention still has the ability to self-repair after experiencing multiple shear thinning, that is, shear thinning can only temporarily destroy the three-dimensional network structure inside the hydrogel, and has the potential to form a gel in situ after injection. Shear thinning can be carried out using a variety of mechanical forces that can apply shear or shear stress to the hydrogel, such as blowing, centrifugation, shaking, injection, spraying, filtering, etc. Furthermore, the advantage of the hydrogel of the present invention is that the self-repair process after shear thinning can be terminated by dilution, that is, after the hydrogel is shear thinned under mechanical force, dilution with a solvent can make it unable to recover to a gel state, but present a liquid state, which is conducive to separating the substances contained in the hydrogel. For example, after cells are cultured and stored and transported with the hydrogel, the cells can be separated from the hydrogel by shear thinning or sufficient dilution to harvest the cells.

[0166] The hydrogel of the present invention can self-assemble without introducing exogenous substances, which means that the hydrogel of the present invention can avoid changing the existing use environment as much as possible when used, and then be used for tissue repair, in vitro 3D cell culture, storage of cells and viruses, cell migration test, organoid construction, preparation of wound dressings, preparation of scaffolds required for tissue engineering and other purposes; and support the use of injection; when used in vitro, it is conducive to the replacement of cell culture and the separation of cells; when used in vivo, it can achieve in situ repair in the body and in vivo drug delivery, etc. For in vitro 3D cell culture, there is no need to add other components other than the complete cell culture medium; for some immune applications, it can avoid introducing other immunogenic components to interfere with the experiment. At the same time, the hydrogel of the present invention will not change the temperature and pH of the entire system during the self-assembly process and / or after the gel is formed, which brings great safety guarantees for its application in the field of biomedicine and can be widely used in the field of biomedicine.

[0167] In some embodiments, the hydrogel of the present invention is used for three-dimensional cell culture. After the self-assembling peptide solution of the present invention is directly mixed with the complete cell culture medium containing cells, it is transferred to a culture dish, a culture bottle, a cell culture plate and other culture equipment to achieve three-dimensional cell culture. The external environmental conditions and culture medium required for three-dimensional culture are consistent with traditional two-dimensional cell culture, and no expensive equipment is required. The hydrogel of the present invention is suitable for three-dimensional culture of various types of cells.

[0168] In some embodiments, the hydrogel of the present invention is used for organoid construction. The self-assembling peptide of the present invention can stably support cells to form a 3D distribution, and then form a functional structure with a preliminary organ morphology under the action of a special culture medium for organoids, which can be used for the cultivation of primary tumor cells, induced pluripotent stem cells, embryonic stem cells and other stem cell differentiation organoids, and the cultivation of other primary cells except tumors. It can be used for drug screening, animal model construction and other applications.

[0169] Glossary:

[0170] Hydrogel is a hydrophilic polymer material that can form a three-dimensional network structure by chemical or physical crosslinking. Hydrogel materials can come from natural and synthetic sources. Natural polymers such as chitosan, alginate, hyaluronic acid (HA), collagen, gelatin, etc. have the advantages of being biodegradable and carrying integrin binding sites, but they are immunogenic. Synthetic polymers such as polyethylene glycol (PEG), polyacrylamide (PAM), polyvinyl alcohol (PVA) and polymethyl methacrylate (PMMA) have the advantages of strong mechanical properties, customizability, and low immunogenicity, but lack innate biological functions and must undergo important post-processing to induce the desired response in vivo.

[0171] Hydrogels have the following properties:

[0172] 1. Good biological relevance: The polymer contains a large number of hydrophilic groups, which can absorb dozens of times more water than itself, and has the characteristics of swelling but not dissolving in water, and has good water retention capacity;

[0173] 2. Similar to the extracellular matrix: Through structural design, the physical, chemical and mechanical properties of the hydrogel can be made similar to those of the extracellular matrix, which is conducive to cell growth and reproduction;

[0174] 3. Biodegradability: Some natural polymer materials are biodegradable, which can avoid secondary damage caused by implant removal. It is these unique advantages that make hydrogels shine in biomedical materials.

[0175] In the present invention, when a peptide having a structure having both a hydrophilic surface and a hydrophobic surface self-assembles under the conditions described in the present invention, especially physiological conditions, a hydrogel is obtained by encapsulating water.

[0176] Peptide Self Assembly is a short chain of amino acids with polar domains. When dissolved in neutral solvents and physiological salt concentrations, these self-assembling peptides spontaneously assemble into hierarchical nanostructures through hydrogen bonds, ionic bonds, hydrophobic interactions or van der Waals forces. Materials derived from these assemblies have the advantages of being non-toxic, non-immunogenic, non-thrombotic, degradable and easily metabolized. At the same time, nanofibers have the same size scale as natural ECM fibers, can be easily designed to simulate the stiffness of various soft tissues, and can be further functionalized by the attachment of cell-interacting peptide domains or cytokines and growth factors. Therefore, they can be used to design biologically relevant culture environments and improve the control of proliferating cell populations.

[0177] Self-assembling peptides can be used as hydrogels for wound treatment as drug carriers, and can also be used as polypeptide nanofibers for cancer treatment to continuously release small molecules, growth factors, and monoclonal antibodies. For example, self-assembling peptides can be used to promote angiogenesis in regenerated tissues and to repair skin wounds using polypeptide-based scaffolds. Therefore, self-assembling peptides can be used in the field of injectable therapeutic hydrogels.

[0178] The alternating hydrophilic and hydrophobic amino acid residues in the self-assembling peptides enable the self-assembling peptides to retain a large amount of water to form a hydrogel. The side chains of the hydrophilic residues can directly interact with water, and the water molecules form inclusion complexes to surround the side chains of the hydrophobic residues. The number of hydrophobic residues and hydrophilic residues in the self-assembling peptides requires clever design and proportioning. If there are too many hydrophobic residues, the self-assembling peptides will be insoluble in water and precipitate out of the water; on the other hand, if there are too many hydrophilic residues, the self-assembling peptides are highly water-soluble and therefore cannot form a hydrogel. In addition, it is also necessary to precisely and cleverly induce the peptide molecules to self-assemble into ordered nanostructured materials, such as nanofibers, nanotubes, and nanovesicles.

[0179] "Amino acid" includes those naturally occurring as well as non-naturally occurring amino acids, such as D-natural amino acids, beta and gamma derivatives. According to standard terminology, amino acid residue sequences can be named using three-letter or one-letter codes, for example: alanine (Ala, A); arginine (Arg, R); asparagine (Asp, N); aspartic acid / aspartic acid (Asp, D), cysteine ​​(Cys, C); glutamine (Gln, Q); glutamic acid (Glu, E); glycine (Gly, G); histidine (His, H), isoleucine (Ile, I); leucine (Leu, L), lysine (Lys, 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); hydroxyproline (Hyp, O).

[0180] In the present invention, a "peptide" is an amino acid chain. In particular, a peptide is 2 to 40 amino acids in length.

[0181] In the present invention, "self-assembly" refers to the aggregation of self-assembling peptides under normal environmental conditions to form an ordered structure.

[0182] In the present invention, "β-fold" refers to a relatively extended periodic folded zigzag main chain conformation in a polypeptide chain, which is arranged in parallel or anti-parallel to form a β-fold (sheet). The parallel or anti-parallel conformation is determined based on the direction of the peptide arrangement from N to C terminus. Parallel arrangement means that the peptide chains are arranged from N to C terminus. Anti-parallel arrangement means that the peptide chains are arranged in opposite directions (i.e., the first peptide chain is arranged from N to C terminus, and the opposite second peptide chain is arranged from C to N terminus). The parallel arrangement may include the two ends of the peptide being staggered with each other due to peptide translation. At least half of the length of the peptide is involved in the interaction force between peptides. In the anti-parallel arrangement, the polypeptides are usually arranged in a line to provide two flush endpoints. This is a typical end-to-end complementary peptide.

[0183] β-turn is an irregular secondary structure in proteins that causes a change in the direction of the polypeptide chain. β-turns often occur at the corner where the peptide chain makes a 180° turn. β-turns are composed of 3-5 amino acid residues, with the second residue being proline (P) or hydroxyproline (O). The β-turn motif generally refers to a turn structure stabilized by hydrogen bonding between the carbonyl oxygen atom of the nth amino acid residue and the amide proton of the n+3th amino acid residue. β-turns are also called β-elbows, reverse elbows or β-loops, and are used to connect β-chains.

[0184] The term "hydrophobicity" in the present invention refers to a property of tending to repel water or being completely insoluble in water.

[0185] The term "hydrophilicity" in the present invention refers to the property of easily absorbing water and having a strong polar group that easily interacts with water.

[0186] Hydrophilic amino acids, also known as polar amino acids, have polar R groups that can generally form hydrogen bonds with water molecules, so they have a certain affinity for water molecules. Hydrophilic amino acids include: S, T, Y, C, U, N, Q, D, E, O, R, K, H.

[0187] Hydrophobic amino acids, also known as non-polar amino acids, have non-polar R groups and have low or very low affinity for water molecules, but have a high affinity for fat-soluble substances. They include: G, A, V, L, I, P, M, F, and W.

[0188] "Nanostructure" refers to a structure with nanometer size. Nanostructures can be any shape in one, two, or three dimensions, including nanofilms, nanofibers, nanorods, nanowires, nanofiber networks, nanospheres, nanohelices, and mixtures thereof. The surface of a nanostructure has a one-dimensional structure at the nanoscale, i.e., the surface thickness of the object is between 0.1nm and 100nm. Nanotubes have two dimensions of nanometer size, with a diameter of 0.1 to 100nm and a length that may be larger. Spherical nanoparticles have three dimensions in nanometer size, i.e., the size of the particle nanoparticle in each spatial dimension is 0.1 to 100 nanometers.

[0189] When the protein solution is at a certain pH, the protein has an equal tendency to dissociate into positive and negative ions, that is, it becomes a zwitterionic ion with a net charge of zero. The pH of the solution at this time is called the isoelectric point (pI) of the protein.

[0190] In the present invention, "response" and "trigger" are both used to indicate that the self-assembling peptides respond to different pH values ​​to form hydrogels, or that environmental factors trigger the responsive self-assembling peptides to form hydrogels, and the two have the same meaning.

[0191] The RGD sequence is a tripeptide sequence containing arginine (R)-glycine (G)-aspartic acid (D). It is the recognition site of integrin and its recognition protein and is widely present in organisms. The adhesion proteins in the extracellular matrix (ECM) and blood are the most common proteins containing the RGD sequence in the human body, mainly including fibrin, laminin, collagen, etc. The RGD sequence mediates the adhesion between cells and the extracellular matrix and between cells, and also has a signal transduction function, thereby mediating many important life activities.

[0192] Microcarriers are usually non-toxic, non-rigid, uniform-density microspheres or microslides used in cell culture. They enable cells that rely on adhesion to grow as a monolayer on the surface of the particles during suspension culture, thereby increasing the area for cell attachment and growth, which is conducive to large-scale cell culture and collection. Microcarriers can play a huge role in the field of large-scale cell proliferation in vitro. According to the shape of the microcarrier, it can be mainly divided into spherical and lamellar, with a size of about 100-500μm. The raw materials for preparing microcarriers include glass, polystyrene plastic, acrylamide, dextran, gelatin, sodium alginate, polylactic acid, chitosan, etc. The surface can be modified to carry a charge; the density is generally controlled at 3%-4% higher than water or lower, and it can be suspended with just a slight stir, and solid-liquid separation can be achieved by cheap methods such as centrifugation or sedimentation. Therefore, currently common commercial microcarriers can be divided into cross-linked dextran microcarriers, cellulose microcarriers, protein microcarriers, and microcarriers based on synthetic polymers according to the raw materials. Among them, cross-linked dextran microcarriers are represented by the Cytodex series, which are surface-modified with denatured collagen or diethylamineethyl (DEAE), and are positively charged as a whole, which is conducive to cell adhesion. They are a type of microcarrier with a wide range of applications; cellulose microcarriers are represented by Cytopore, which are positively charged through DEAE modification and have a porous structure. In addition to providing surface area for cell adhesion, they can also protect cells from shear damage; protein microcarriers are mostly based on gelatin or denatured collagen, such as Tantti 3D cell culture scaffolds, which are based on natural collagen and have a three-dimensional macroporous structure, which can be used for in vitro cell amplification culture. In addition to the above-mentioned natural polymers, microcarriers made of other artificial synthetic materials, such as glass, polystyrene, etc., have the characteristics of high reproducibility. However, due to the lack of sites on their surfaces for cell recognition and adhesion, their application is limited. Existing commercial microcarriers are mostly used in the fields of biomedicine and cell culture, and there are few related applications in the food industry. When selecting microcarriers for in vitro cell culture, in addition to the effects of conventional parameters such as density and surface properties on cell attachment and proliferation, special requirements must be placed on detachability or degradability. Otherwise, additional separation steps are required after the culture is completed, increasing the complexity of the process and production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0193] Figure 1 The macroscopic rheological states of the self-assembling peptide of the present invention (SEQ ID NO: 2 as an example) under different pH conditions are shown: A: pH = 2; B: pH = 6; C: pH = 6.4; D: pH = 7; E: pH = 9; F: pH = 12.

[0194] Figure 2 The macroscopic rheological state of 1.0 wt % of the self-assembling peptide of the present invention (SEQ ID NO: 2 as an example) after responding to 3 mM calcium chloride (A), 3 mM magnesium chloride (B), and 1.0 wt % bovine serum albumin (C) under neutral conditions (pH = 7) is shown.

[0195] Figure 3 The rheological properties of a PBS solution containing 2.0 wt % of SEQ ID NO: 30 (A) and a PBS solution containing SEQ ID NO: 2 (B) under neutral conditions (pH=7) are shown.

[0196] Figure 4 TEM images of self-assembling peptides of SEQ ID NO: 1 (A), SEQ ID NO: 13 (B), and SEQ ID NO: 22 (C) are shown.

[0197] Figure 5 The microstructure of the freeze-dried matrix of SEQ ID NO: 24 at concentrations of 1 wt % (A), 2 wt % (B), and 3 wt % (C) observed by scanning electron microscopy (SEM) is shown.

[0198] Figure 6 The rheological states of SEQ ID NO: 25 in deionized solution at pH=3, pH=6.7, and pH=12 are shown.

[0199] Figure 7 The infrared spectra of SEQ ID NO: 25 in deionized solution (A) at pH=3, pH=6.7, pH=12 and PBS buffer, α-MEM medium containing 10% calf serum and pure calf serum (B) are shown.

[0200] Figure 8 The fluorescence spectrum responses of thioflavin at concentrations of 100 μM, 200 μM, and 400 μM of SEQ ID NO: 29 are shown.

[0201] Fig. 9 The supporting function of SEQ ID NO:2 and SEQ ID NO:3 on red blood cells is shown; the four parallel vials are 0.1wt%, 0.05wt%, 0.01wt% of 2mL of pure aqueous solution of self-assembling peptide (containing 10% calf serum and 10 8 of red blood cells) and a blank group (containing no self-assembling peptide solution, 10% calf serum and 10 8 of red blood cells).

[0202] Fig.10 The rheological properties of 0.3wt% (A), 0.5wt% (B), and 1wt% (C) of the self-assembling peptide stock solution of SEQ ID NO: 10 in DMEM basal medium are shown, wherein G' is the storage modulus and G" is the loss modulus.

[0203] Fig.11The rheological properties of 0.3 wt % (A), 0.5 wt % (B), and 1 wt % (C) of the self-assembling peptide stock solution of SEQ ID NO: 10 in DMEM complete medium containing 10% calf serum are shown.

[0204] Fig.12 The storage modulus (G') at 1500 minutes after 0.5 wt % of SEQ ID NOs: 1 to 30 was mixed with DMEM complete medium containing 10% calf serum is shown.

[0205] Fig.13 The injectability verification of 2 wt % of the self-assembling peptide of SEQ ID NO: 20 in PBS solution is shown.

[0206] Fig.14 The shear stress and self-repair characteristics of 1 wt % SEQ ID NO: 24 self-assembled into gel at pH = 6.4 are shown.

[0207] Fig.15 The changes in storage modulus (G') and loss modulus (G") of 0.3 wt.%, 0.5 wt.%, and 1 wt.% SEQ ID NO: 9 self-assembling peptide solutions at different frequencies are shown.

[0208] Fig.16 Zeta potential detection and pH response of the self-assembling peptide of SEQ ID NO: 29 are shown.

[0209] Fig.17 The SEM image of a 0.5 wt % solution of the self-assembling peptide of SEQ ID NO: 24 forming a stable hydrogel after freeze-drying is shown.

[0210] Fig.18 It shows that a 0.5 wt % solution of the self-assembling peptide of SEQ ID NO: 24 forms a stable hydrogel and then liver cancer cells are cultured on the hydrogel.

[0211] Fig.19 The supporting effects of SEQ ID NO: 9 on liver cancer cells at final concentrations of 0 (control), 0.1wt%, 0.3wt%, and 0.5wt% were shown.

[0212] Fig. 20 The effect of (2D) and (3D) the self-assembling scaffold solution of SEQ ID NO: 22 on cell survival of mouse mesenchymal stem cells during refrigerated storage is shown.

[0213] Fig.21It shows that adding 0.1wt.%, 0.3wt.%, and 0.5wt.% of the SEQ IDNO:9 self-assembling peptide to the cell culture system, respectively, induces its self-assembly into a fiber network scaffold and cultures liver cancer cells. The cell sphere diameters are counted on the 3rd day, 7th day, 11th day, and 15th day, respectively, and the characteristic cell spheres in the culture system are photographed.

[0214] Fig. 22 The images are shown after the cultured liver cancer cells were stained with Calcein AM and then imaged under a laser confocal microscope. A is Calcein AM live-dead staining, and B is EDU staining.

[0215] Fig.23 The self-assembled mesh scaffold of SEQ ID NO: 21 was used to culture porcine muscle satellite cells under liquid conditions.

[0216] Fig.24 The cell morphology of primary esophageal cancer organoids cultured with SEQ ID NO: 21 after 7 days is shown.

[0217] Fig.25 The figure shows the supporting effect of the self-assembling peptide solution of SEQ ID NO: 23 on polylactic acid microspheres (A) and polystyrene microspheres (B). The left figure in A and B is the microsphere solution without adding 0.1wt% of the self-assembling peptide, and the right figure is the microsphere / self-assembling peptide solution with adding 0.1wt% of the self-assembling peptide.

[0218] Fig.26 The figure shows that pig muscle satellite cells cultured in the polylactic acid microsphere / self-assembling peptide composite system were stained with Calcein-AM / PI dye and then detected using a laser confocal microscope. A, B, C, and D represent cells on the 1st, 3rd, 5th, and 7th days, respectively.

[0219] Fig. 27 The figure shows that porcine muscle satellite cells cultured in the polystyrene microsphere / self-assembling peptide composite system were stained with Calcein-AM / PI dye and then detected using a laser confocal microscope. A, B, C, and D represent cells on the 1st, 3rd, 5th, and 7th days, respectively.

[0220] Fig.28 The cell proliferation curve and cell viability characterization results of porcine muscle satellite cells cultured in the polylactic acid microsphere / self-assembling peptide composite system are shown.

[0221] Fig.29 The cell proliferation curve and cell viability characterization results of porcine muscle satellite cells cultured in the polystyrene microsphere / self-assembling peptide composite system are shown.

[0222] Fig.30Crystal violet stained micrographs of HepG2 and Hela cells growing adherently on a plate after migration are shown.

[0223] Fig.31 The images show the livers engrafted with tumors in situ and dissected out from the experimental group (A) and the control group (B) after 18 days of culture.

[0224] Fig.32 The figure shows the comparison of tumor proliferation between the experimental group and the control group after N87 cells were implanted subcutaneously in SCID Beige mice. A shows the change in tumor volume, and B shows the photos of the experimental group and the control group mice on the 18th day.

[0225] Example

[0226] The present invention is further explained below through specific examples. Since the principle of self-assembly of self-assembly peptides containing RGD to form a three-dimensional network material is consistent with the present invention, in multiple embodiments, different self-assembly peptide sequences are used as examples to provide verification data on the hydrogel properties and functions of the present invention. It is understandable that other self-assembly peptides that conform to the molecular structure of the self-assembly peptide of the present invention, for which detailed data are not provided in the present invention, also have the same or similar effects. The amino acids mentioned in the present invention are characterized by single-letter abbreviations, which are generally accepted in the technical field.

[0227] Example 1. Synthesis of self-assembling peptides

[0228] The self-assembling peptide of the present invention is synthesized by a standard solid phase peptide synthesis method. The amino acid sequence of the self-assembling peptide is shown in SEQ ID NO: 1-31 in the sequence table. It is confirmed to be the target self-assembling peptide by high performance liquid chromatography and mass spectrometry. The self-assembling peptide powder is obtained after freeze drying and stored at -20°C for drying.

[0229] Example 2. Preparation of self-assembling peptide solution

[0230] Weigh an appropriate amount of self-assembling peptide and dissolve it with deionized water. If there is still a small amount of undissolved, add a small amount of alkaline solution, such as sodium hydroxide, to dissolve it. After dissolution, test the pH value of the self-assembling peptide solution to make it between 7.0 and 7.2. Prepare a 2wt% self-assembling peptide mother solution, sterilize it with high pressure, and refrigerate it at 4°C. Prepare enough mother solution to ensure that the same batch of self-assembling peptides are used in the same experiment.

[0231] Example 3: pH, metal ion and protein response verification of self-assembling peptides

[0232] Experimental method: Prepare 2wt% and 1wt% self-assembling peptide solutions under different pH environments: Taking SEQ ID NO:2 as an example, add deionized water to dissolve the lyophilized powder and prepare a 2wt% self-assembling peptide mother solution; use sodium hydroxide and hydrochloric acid to adjust the pH so that the concentration of the self-assembling peptide solution is 1wt%, and the pH values ​​are 2, 6, 6.4, 7, 9, and 12, respectively. After standing for 24 hours, observe and take pictures.

[0233] When the self-assembling peptide solution was neutral (pH = 7), the response of the self-assembling peptide solution to metal ions (such as magnesium ions and calcium ions) and proteins (such as BSA) was tested. Taking SEQ ID NO: 2 as an example: deionized water was added to dissolve the lyophilized powder, and sodium hydroxide and hydrochloric acid were used to adjust the pH value of the self-assembling peptide solution to 7 and a concentration of 2wt%. An equal amount of 6mM magnesium chloride or calcium chloride, or an equal amount of 2wt% BSA aqueous solution was added, respectively, so that the concentration of the self-assembling peptide in the final system was 1wt%, the concentration of the metal ion was 3mM, or the concentration of BSA was 1wt%. After standing for 24h, the system was observed and photographed.

[0234] Experimental results: When the concentration of the self-assembling peptide is 1%, it is liquid under acidic conditions (pH = 2 and 6); it is a gel peptide at pH 6.4 and does not flow when placed tilted; it is a relatively viscous liquid at pH 7.0, and it is liquid at pH = 9.0 and pH = 12.0 (as shown in Figure 1). Figure 1 AF). By comparison, it was found that: SEQ ID NO: 4 was liquid under acidic conditions (pH = 3); it was liquid at pH 6.0, and the viscosity was enhanced; it was liquid at pH 12.0, and had a certain viscosity; while SEQ ID NO: 5 was liquid in all pH solutions; the 10th amino acid in SEQ ID NO: 5 (missing the β-turn region) was O, and the amino acid in the previous position was isoleucine, and the sequence basis for forming the β-turn region was completely destroyed. This example proves that the β-turn region is a necessary condition for the self-assembly of the self-assembly peptide of the present invention; while SEQ ID NO: 30 (missing the RGD sequence) was colloid at pH 2, 6, and 6.4, and the other pH environments were liquid, which verified that the RGD sequence was the second necessary condition to ensure self-assembly under weak acid and neutral conditions; while SEQ ID NO: 31 (missing the RGD sequence) was a turbid solution state and could not be completely dissolved, which again proved that RGD participated in the function of the self-assembly peptide of the present invention.

[0235] In the metal ion and protein responsiveness test of the polypeptide sequence of SEQ ID NO: 2, it was found that when 3 mM magnesium ions and 1 wt % BSA were added to a 1 wt % self-assembling peptide solution at a neutral pH of 7, the solution did not start to form a significant hydrogel within 30 minutes; however, when calcium ions were added, the self-assembling peptide rapidly self-assembled and immediately became a weaker hydrogel (e.g. Figure 2 AC in FIG). By comparing SEQ ID NO:30, it was found that the above factors can all activate SEQ ID NO:30 to form a stable hydrogel. The difference between SEQ ID NO:2 and SEQ ID NO:30 is that the amino acid sequence GGE is replaced by the amino acid sequence RGD at the end of the hydrophilic end of the polypeptide in SEQ ID NO:2. This test proves that the replacement can significantly change the self-assembly properties of the polypeptide. By comparing SEQ ID NO:2 and SEQ ID NO:31, it was found that when the amino acid sequence RGD was missing (SEQ ID NO:31), the polypeptide was turbid under all test conditions and could not cause a response. This experiment once again proves the important role of RGD in the self-assembly function of the polypeptide.

[0236] Even when the concentration of SEQ ID NO:30 in PBS buffer was increased to 2 wt %, the mixed solution still maintained good fluidity and appeared liquid ( Figure 3 A), when the concentration of the self-assembling peptide of the present invention in PBS buffer is increased to 2 wt%, the mixed solution presents a gel with relatively high elasticity and viscosity ( Figure 3 B) This verification once again proves that the RGD sequence completely changes the self-assembly properties of the peptide without the RGD sequence. That is, the peptide of the present invention containing RGD can no longer respond to the obvious triggered assembly of metal ions and proteins, but can self-assemble itself.

[0237] The responses of the self-assembling peptides shown in each sequence to different pH, metal ions and proteins are shown in Table 1.

[0238]

[0239] Experimental conclusion: Figure 1As shown in the results of Table 1, the self-assembling peptide hydrogel exhibits a reversible solution-gel transition in response to a single stimulus of pH change. The self-assembling peptide can self-assemble under neutral, i.e. physiological pH conditions to cause macroscopic gelation of the system, demonstrating its potential application value as a network scaffold material with a supporting function in a physiological environment. The hydrophobic region of SEQ ID NO:4 consists of four hydrophobic amino acids, which are in a liquid state with a certain viscosity at a pH value of 7.0-9.0, which reflects that the hydrophobic sequence of the polar peptide still has a certain self-assembly function when it contains four hydrophobic amino acids. The difference between SEQ ID NO:5 and other polypeptides is that the 10th amino acid is proline and the 9th amino acid is isoleucine. This sequence feature destroys the conditions for the SEQ ID NO:5 polypeptide to form a β-turn, so the SEQ ID NO:5 polypeptide loses its self-assembly function. Therefore, it is found by comparison that the self-assembling peptide that cannot form a β-folded structure cannot trigger the formation of a hydrogel (such as SEQID NO:5); SEQ ID NO:30 has two β-turns, and SEQ ID NO:31 only contains one β-turn. Both polypeptides lack the RGD sequence, but they show completely different rheological properties. SEQ ID NO:30 can respond to acidic pH, magnesium ions, calcium ions, proteins, etc., but cannot self-assemble under neutral conditions without a starting component; while SEQ ID NO:31 becomes a polypeptide with very low solubility under various pH conditions and completely loses the ability to self-assemble. These verifications fully verify the necessity and importance of the β-turn and RGD sequence for the self-assembling peptide of the present invention to play a specific function.

[0240] Example 4: Structural characterization and self-assembly mechanism study of mesh scaffolds

[0241] Experiment 1: Transmission Electron Microscopy (TEM)

[0242] Experimental method: Preparation of self-assembling peptide solution for TEM experiment: The 0.2 wt % self-assembling peptide solution obtained in Example 2 was diluted with ultrapure water to a self-assembling peptide concentration of 0.002 wt %.

[0243] Preparation of self-assembling peptide samples: Dilute the 0.2wt% self-assembling peptide solution obtained in Example 2 with ultrapure water to 0.002wt%, incubate at 37°C for one hour, take 10μL and place it on a 300-mesh carbon support film copper mesh (purchased from Beijing Xinxing Bairui Technology Co., Ltd.), and let it stand at room temperature until it is completely dry. After complete drying, use 10μL of 2wt% phosphotungstic acid negative staining solution to drop on the copper mesh carrying the sample, absorb the excess stain after staining for 60 seconds, and repeat this staining step three times. After staining, let the copper mesh stand at room temperature to dry.

[0244] Experimental results: TEM can better see the secondary structure of the self-assembling peptide after assembly. All samples were observed and photographed using a TalosG2 200X transmission electron microscope. Taking SEQ ID NO:1, SEQ ID NO:13, and SEQ ID NO:22 as examples, the imaging results are as follows: Figure 4 As shown: Figure 4 A, B, and C are TEM images of self-assembling peptides with a concentration of 0.002wt% of SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 22. The TEM can observe the mesh structure inside the self-assembling peptide. It can be seen from the figure that the self-assembling peptide of the present invention can be self-assembled at low concentrations without an excitation substance. The nanofiber length is several hundred nanometers and the width is about 4nm, and the self-assembling peptide itself is in the shape of a short ribbon. The self-assembling peptide of the present invention should be a concentration-responsive self-assembling peptide. As the concentration changes, its own structure is found to change significantly. The above phenomenon shows that under physiological conditions, the self-assembling peptide will undergo stable and orderly self-assembly, thereby forming a more dense, stable, strong and uniformly porous nanofiber three-dimensional network structure, which can efficiently retain moisture and form a structure with good support. This structure is conducive to supporting cells during cell culture and building a three-dimensional growth space for cells.

[0245] The results of the experiments of the self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0246] Experiment 2: Scanning Electron Microscopy (SEM)

[0247] Experimental method: The self-assembling peptide powder prepared in Example 1 was diluted with complete culture medium (DMEM high-glucose culture medium with 10% fetal bovine serum and 1% double antibody), and the dilution concentration was selected as the preparation of the self-assembling peptide solution for SEM experiment: The self-assembling peptide solution obtained in Example 2 was diluted with ultrapure water to a concentration of 1wt%, 2wt%, and 3wt%. After dilution, it was first placed in a deep low-temperature refrigerator at -80°C to make it completely frozen, and then frozen under liquid nitrogen for 15 minutes, and then a vacuum freeze dryer was used for 24h low-temperature vacuum complete freeze drying. After freeze drying, it was photographed under a scanning electron microscope (JSM-IT300LV) as the experimental group, and the control group was the SEM image of the self-assembling peptide itself. Before observation, gold needs to be sprayed on the surface of the sample to increase the conductivity of the sample. At the same time, the aperture size statistics of the photographed pictures were performed by Image J.

[0248] Experimental results: 1wt%, 2wt%, and 3wt% samples all exhibited a porous structure. In the complete culture medium, the self-assembling peptides were interconnected and highly connected, forming a honeycomb-like porous structure. This structure is suitable for the living environment of cells and can achieve the transportation of nutrients and oxygen. At the same time, the self-assembling peptides of the present invention contain RGD (arginine-glycine-aspartic acid) sequences, which exist in various extracellular matrices and can effectively promote the adhesion of cells to biomaterials. One of the conditions required for cell growth is a suitable pore size. On the other hand, large pores with a high surface area can accommodate cells and cell spheroids. Therefore, the structure formed by the self-assembly of the self-assembling peptides of the present invention is more suitable for cell growth. At the same time, as the concentration of the self-assembling peptides increases, the pore size of the mesh scaffold becomes smaller, and the structure becomes more regular. Figure 5 As an example, the microstructures of the freeze-dried matrix of SEQ ID NO: 24 at concentrations of 1% (A), 2% (B), and 3% (C) are presented respectively.

[0249] Experiment 3: Infrared (FTIR) analysis

[0250] Experimental method: Taking SEQ ID NO: 25 as an example, dissolve the self-assembling peptide in deionized water, prepare 1mL of 2wt% self-assembling peptide solution and sterilize. Take 100μL of self-assembling peptide solution, adjust the pH to 3, 6.7, and 12 with NaOH and HCl, respectively, and adjust the concentration of self-assembling peptide to 1.0wt% with deionized water; take 100μL of self-assembling peptide solution, add 100μL PBS, 100μL α-MEM, 100μL α-MEM containing 10% FBS, and 100μL deionized water containing 20wt% fetal bovine serum (FBS), respectively, and the pH is 7.2 after testing. The total volume of the sample is 200μL, the final concentration of the self-assembling peptide is 1wt%, and it is freeze-dried after quick freezing in liquid nitrogen for 15min. FT-IR spectroscopy was used to confirm the presence of hydrogen bonds and determine the protonation state of the carboxylic acids. Spectra of samples prepared by the KBr pellet method were obtained using a Nicolet iS5 FTIR-IR spectrometer in attenuated total reflectance (ATR) mode with a wavenumber scan range of 1750–1450 cm -1 The wave number resolution is 0.5 cm -1 The infrared data is normalized using origin.

[0251] Experimental results: The states of SEQ ID NO:25 in deionized solution at pH 3, 6.7, and 12 were respectively liquid, gel peptide, liquid ( Figure 6 ). From the infrared image ( Figure 7 As can be seen from Figure AB), the self-assembling peptide solution at pH = 6.7 has a higher peak at 1670 cm-1 than the self-assembling peptide solution at pH = 3 or 12. -1Most of the β-rotation structure peaks disappear at 1634 cm -1 The peak at 1634 cm is sharper, indicating that more β-folded structures have been formed. -1 ); and the infrared images when stimulated by PBS, α-MEM (10% FBS), and FBS respectively show that PBS and α-MEM (10% FBS) have similar effects on the secondary structure, and the self-assembling peptide stimulated by 10% serum alone has little effect on the secondary structure. This study proves that in the process of the self-assembling peptide of the present invention forming a mesh scaffold, the β-turn structure plays a vital role in the self-assembly, and the β-turn angle provides a favorable basis for forming more β-folding structures and making the fiber structure more regular and orderly.

[0252] Experiment 4: Thioflavin fluorescence experiment

[0253] Experimental method: Taking SEQ ID NO:29 as an example, 5mM THT dye solution was diluted to 0.1mM with ultrapure water, and self-assembling peptide solutions with concentrations of 200μM, 400μM, and 800μM were prepared with ultrapure water. The self-assembling peptide solution was mixed with the diluted dye solution at a ratio of 1:1 to make the final self-assembling peptide concentrations of 100, 200, and 400μM. After mixing, it was incubated in an oven at 37°C for 1h. At the same time, the fluorescence spectrophotometer was turned on 30min in advance, and the parameters were set. The excitation wavelength was 442nm, the emission wavelength was 450-550nm, and the voltage was 800V. Five spectra were collected for each sample, with an excitation wavelength of 442nm, and the excitation and emission slits were 5nm and 2.5nm, respectively. Each group was repeated 5 times, and the average value was calculated. After the measurement, Origin was used for data analysis.

[0254] Experimental results: As a potential dye for quantitative analysis of the β-folding structure in self-assembling peptide nanofibers, thioflavin T (ThT) combined with fluorescence can reflect the existence of β-folding in the solution. For this reason, different concentrations of self-assembling peptides were selected for experiments to change the number of nanofibers. Figure 8 The figure shows the self-assembling peptide after ThT fluorescence staining. The final self-assembling peptide concentrations selected were 100, 200, and 400 μM. When the emission wavelength was between 450-550 nm, the peak value of ThT binding fluorescence changed with the change of peptide concentration. β-folds existed in the self-assembling peptide gel, and their content was positively correlated with the concentration of the self-assembling peptide, indicating that the self-assembling peptide was a concentration-dependent self-assembling peptide. This is because the increase in peptide monomers makes it easier for molecules to contact, thereby increasing the degree of assembly. The gel ThT staining data showed that under neutral pH conditions, the supramolecular polymers generated by the self-assembling peptide exhibited a typical β-fold structure. This experiment once again demonstrated the importance of β-folds formed by β-turns in the polypeptide structure for the self-assembly function of polypeptides.

[0255] Example 5: Effects of amino acid sequence structure and initiating components on the physical and functional properties of the peptide self-assembly material of the present invention, and verification of the self-assembly mechanism

[0256] Experiment 1: Verification of amino acid sequence support for red blood cells

[0257] In this experiment, red blood cells were used as an example to verify the supporting effect of amino acid sequences on cells, especially the supporting effect of hydroxyproline in the amino acid sequence on cells.

[0258] Experimental method: PBS buffer was used to prepare 0.1 wt % of the self-assembling peptide mother solutions of SEQ ID NO: 2 (IIIIIGSIIGPGGEGPRGD) and SEQ ID NO: 3 (IIIIIGSIIGOGGEGPRGD), respectively, and sterilized at high temperature.

[0259] Using a 2 mL transparent glass bottle, 2 mL of pure self-assembling peptide aqueous solution (containing 10% calf serum) and a blank group (containing no self-assembling peptide solution and 10% calf serum) were prepared with 0.1 wt%, 0.05 wt%, and 0.01 wt% of the two materials, respectively. 9 The final concentration of red blood cell stock solution was 1×10 8 cells / mL, blow evenly. Take pictures every 4 hours to compare the supporting effects of different materials on red blood cells. Since the hydrogel formed by the self-assembling peptide of the present invention is a water-swelling system, the low-concentration self-assembling peptide network scaffold, although in a solution state, still reflects the supporting ability of the self-assembling peptide hydrogel.

[0260] Experimental conclusion: Among the self-assembling peptides with similar amino acid sequences, hydroxyproline has a significant effect on the cell-supporting capacity of the hydrogel or the scaffold solution formed by the swelling of the hydrogel. Fig. 9 As shown in the figure, under low concentration conditions (0.01wt%), the red blood cell supporting capacity of the two mesh scaffolds showed obvious sedimentation within 4 hours. Under high concentration conditions (0.1wt%, 0.05wt%), both self-assembling peptide solutions can effectively support a concentration of 1×10 cells / mL. 8of red blood cells; the self-assembling peptide solution of SEQ ID NO:3 (IIIIIGSIIGOGGEGPRGD) showed no obvious sedimentation and stratification at 8 hours, 24 hours, 32 hours and 48 hours respectively; and although the self-assembling peptide solution of SEQ ID NO:2 (IIIIIGSIIGPGGEGPRGD) could still support the three-dimensional distribution of red blood cells, stratification occurred after 24 hours, and the stratification phenomenon became gradually obvious with the extension of time. Hydroxyproline (O) exists in large quantities in the collagen tissue of animals, and can enhance the elasticity and supporting force of the protein matrix through the hydrogen bonds formed by the hydroxyl groups therein. In the present invention, by introducing hydroxyproline, the stability of the β-fold formed by the self-assembly of the self-assembly peptide can be significantly enhanced through the enhanced hydrogen bond network between the self-assembly peptides, thereby improving the stability of the self-assembly scaffold, thereby enhancing the supporting function of the cells, and making the scaffold material more supportive of the cells.

[0261] Experiment 2, rheological experiment: determination of changes in mechanical properties of mesh scaffolds and the influence of amino acid sequence on self-assembly function.

[0262] Experimental method: Preparation of self-assembling peptide solution for dynamic rheological test: Take the self-assembling peptide mother solution prepared in Example 2 with a concentration of 2 wt %.

[0263] Preparation of self-assembling peptide samples: Using the self-assembling peptide shown in SEQ ID NO: 10, the 2 wt % self-assembling peptide stock solution of Example 2 was diluted to 0.3 wt %, 0.5 wt %, and 1 wt % with DMEM basal culture medium containing no or 10% calf serum.

[0264] In order to simultaneously measure the formation speed and strength of the mixed system gel, 200 μL of each group of samples was immediately added to the 20 mm diameter panel of the MARS 60 rheometer as soon as it was prepared. The final modulus was recorded after a 30-minute time scan. The distance between the rotor and the panel was 500 μm, the shear strain was 1%, the frequency was 1 Hz, and the entire test was completed at 37°C. The 2 wt% self-assembling peptide mother solution of Example 2 was mixed with DMEM culture medium containing no or 10% calf serum, and the final concentrations of the self-assembling peptides were diluted to A (0.3%), B (0.5%), and C (1%), respectively. The samples were stored in a 37°C incubator for 30 minutes. The test results are shown in Table 1. Fig.10 and Fig.11 .

[0265] In this example, the storage modulus (G') of SEQ ID NO: 1-30 polypeptides at a concentration of 0.5 wt% after being stored in a 37° C. incubator for 1500 minutes was tested and compared. The test results are shown in Fig.12 .

[0266] Experimental results: In the samples with different concentrations of self-assembling peptide, when the self-assembling peptide exists alone, its storage modulus is higher than the loss modulus, and the macroscopic appearance is a relatively viscous hydrogel state, while the rheological data at different concentrations show that the storage modulus is less than 5( Fig.10 After adding calf serum, the storage modulus (G') was significantly enhanced. Even at a lower concentration (0.3%), the scaffold system still showed obvious gelation ( Fig.11 AC). This experiment proves that the mesh scaffold system assembled by the self-assembling peptide of the present invention can be enhanced in response to serum, and the mechanical strength is continuously enhanced over time. This unique property gives the material a very important advantage in 3D cell culture. In 3D cell culture, the concentration of cells will gradually increase, and cell clusters may even appear, and the continuously enhanced mesh scaffold continuously increases the support force for the cells in the process, realizing the 3D distribution of the cell system during the culture process.

[0267] Comparison of the storage modulus of different samples at 0.5wt% in DMEM revealed that ( Fig.12 ), the amino acid sequence has a significant effect on the network structure formed by the self-assembling peptide. SEQ ID NO: 8, 9, 12, 28, 29 have higher storage modulus values, and the common point of these sequences is that alanine replaces glycine in the β-turn region. This result proves that the presence of slightly hydrophobic alanine can significantly enhance the mechanical strength of the self-assembled network structure.

[0268] In contrast, the positions of G and P in SEQ ID NO:5 are reversed, and the structure of the β-turn region is destroyed, which causes SEQ ID NO:5 to lose its self-assembly function, so it has no storage modulus in DMEM complete medium; and due to the absence of the RGD sequence in SEQ ID NO:30, it is also a turbid solution in DMEM complete medium. This experiment verifies that the RGD sequence is involved in the self-assembly of the self-assembling peptide in DMEM complete medium and is an indispensable functional structure in the self-assembling peptide of the present invention.

[0269] Experiment 3: Verification of the injectability of the mesh scaffold solution system

[0270] Experimental method: Taking SEQ ID NO: 20 as an example, prepare 0.5 mL of PBS solution of 2 wt% of self-assembling peptide, adjust the pH to 7.0 with sodium hydroxide, leave at room temperature for 24 hours, and observe that the mixed system has formed a hydrogel. Draw the hydrogel with a 1 mL syringe and quickly inject it back into the bottle to observe the effect of shear on the state of the hydrogel during the transfer process.

[0271] Experimental results: Fig.13As shown, 2wt% of the self-assembling peptide of SEQ ID NO:20 can form a hydrogel with high mechanical strength in PBS without any starting component, proving that the self-assembling peptide of the present invention can self-assemble in a neutral environment to form a mesh scaffold system. The formed hydrogel changes from solid to liquid after being subjected to the shear force of the syringe, proving that the hydrogel has the property of shear thinning, that is, the viscoelasticity can be reduced after being subjected to shear force, so that it has fluidity and is convenient for injection and transfer; after standing for about 10 minutes, the liquid sample in the bottle returns to a solid gel state. It shows that the gel assembled by the self-assembling peptide has a good self-repair function; since the driving force for the self-assembly of the self-assembling peptide of the present invention is the interaction of van der Waals force, hydrophobic force, hydrogen bond, etc., these weak interactions can be quickly recombined after standing for a while, thereby driving the self-assembling peptide to become an ordered structure again, forming a mesh scaffold, and showing the re-formation of the hydrogel on a macro scale.

[0272] Experiment 4: Rheological experiment: Determination of the force self-healing function of the mesh scaffold

[0273] Experimental method: Same as experiment 3, with the self-assembling peptide shown in SEQ ID NO:24, 1.0wt% of the self-assembling peptide dissolved in deionized water, and the pH adjusted to 6.4 with HCl. During the test, 200μL of the mixed system was added to the panel with a diameter of 20mm of the MARS 60 rheometer for time scanning. Then, shear thinning was performed for 20 seconds under the conditions of a distance of 500μm between the rotor and the panel, a shear strain of 100%, and a frequency of 1Hz. After that, shear thinning was stopped, and the shear strain was immediately adjusted to 1%. A time scanning test was immediately performed to monitor the recovery of the mechanical strength of the hydrogel. A 20-second shear thinning test was used as a group of experiments, and the group experiment was repeated twice to test the reassembly ability of the hydrogel of the present invention after being deformed by multiple external forces.

[0274] Experimental results: Fig.14 As shown in the figure, the self-assembled hydrogel exhibits the property of being able to quickly reassemble itself after being deformed by external force, and this property does not disappear with the increase in the number of damages. When subjected to 100% shear strain, some non-covalent bonds inside the hydrogel, such as hydrogen bonds, break, causing its mechanical strength to decrease and turn into a liquid state; when the external force disappears, the partially destroyed nanofiber clusters will quickly approach and reassemble, quickly restoring their original mechanical strength. Fig.14It is shown that after multiple shear thinning, the hydrogel of the present invention can still self-assemble again, and the self-assembly time and the strength of the reconstituted hydrogel are extended with the increase of the number of shear thinning, with only a small loss. This experiment proves that the self-assembling peptide formed by the present invention has the characteristics of shear thinning and self-repair, and can be injected. When the hydrogel of the present invention is applied to cell culture, multiple pipetting transfers can be achieved, which is beneficial for cell packaging and cell fluid replacement; at the same time, when it is applied to the field of biomedicine, multiple injections can be achieved.

[0275] The results of the experiments of the self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0276] Experiment 5: Mechanical stability of the mesh stent of the present invention

[0277] Experimental method: A shear thinning test was performed to verify the reassembly performance of the hydrogel of the present invention after deformation.

[0278] Preparation of hydrogel samples: The 2 wt % self-assembling peptide stock solution of Example 2 was diluted to 0.3 wt %, 0.5 wt %, and 1 wt % with DMEM basal culture medium without or with DMEM basal culture medium containing 10% calf serum.

[0279] Taking SEQ ID NO: 9 as an example, the gel sample was subjected to a frequency scanning experimental analysis using a German HAAKE MARS60 rheometer. The viscoelastic properties of hydrogels of different concentrations and complete culture medium were examined using oscillatory rheology. The oscillation frequency scanning mode was selected. Under a strain of 1%, the frequency was set from 0.1 Hz to 10 Hz, and the temperature was maintained at 37° C. The changes in the storage modulus (G′) and the loss modulus (G″) of the material under the degree of frequency change were recorded.

[0280] Experimental results: In the samples with different concentrations of the self-assembling peptide, even at a higher frequency, the storage modulus is higher than the loss modulus, and the macroscopic performance is a hydrogel state. This experiment proves the stability of the self-assembling peptide self-assembling to form a mesh scaffold. Even when subjected to a high shear force, the hydrogel still exhibits a high mechanical stability (e.g. Fig.15 As shown). Combined with Experiment 2 and Experiment 3 in this example, it is proved that the mesh scaffold formed by the self-assembling peptides of the present invention has both stability and injectability. When the shear force is relatively low, the mesh scaffold formed by the self-assembling peptides is relatively stable, and when subjected to a stronger shear force, the mesh scaffold can be destroyed, but once the shear force stops or weakens, the mesh scaffold can quickly self-repair and function. When subjected to a shear process similar to injection, the self-assembling peptide hydrogel is destroyed and forms a liquid, so that it can smoothly enter the tissue, and after entering the tissue, it can quickly form a stable hydrogel package, and can withstand scenes such as bumps and slight vibrations, so as to function stably.

[0281] The results of the experiments of the self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0282] Experiment 6: Charge detection of the self-assembly structure of the self-assembling peptide of the present invention

[0283] Experimental method: Taking SEQ ID NO: 29 as an example, at room temperature, the self-assembling peptide sample was dissolved in ultrapure water to a final concentration of 0.25wt.%, and appropriate amounts of 1M HCl and 1M NaOH were added to make the pH values ​​reach 2.0, 4.0, 6.0, 7.0, 8.0, 10.0, and 12.0, respectively. The points where the pH value suddenly changed were marked as protonation and deprotonation points. The zeta potential of the concentrated self-assembling peptide solution was measured using a zeta potential meter.

[0284] Experimental results: The main self-assembly principle of the self-assembling peptide of the present invention is to control the weak interactions such as hydrophobic force, hydrogen bond, van der Waals force, ionic bond, etc. between the self-assembling peptide molecules in the solution, drive them to form a mesh scaffold structure at their physiological pH value, and then produce various functional applications of the self-assembling peptide. In the solution, positively charged hydrogen ions or negatively charged hydroxyl groups neutralize oppositely charged amino acid residues through electrostatic action, thereby reducing the electrostatic repulsion between peptide molecules, enhancing the interaction between peptide molecules and initiating self-assembly. The positive and negative Zeta potential detects what kind of charge the bio-based macromolecule carries. When the charge of the amino acid residues close to the peptide molecule is neutralized, the Zeta potential will decrease. Fig.16 As shown, when the overall environment is acidic, the carboxylic acid of the self-assembled peptide chain is uncharged, and NH2 binds to one H + Formation of NH3 + Positively charged. Because the self-assembled peptide chain is positively charged, it cannot form a gel under electrostatic repulsion. When the NaOH solution is added gradually to the alkaline environment, COOH gradually loses a proton to form COO - , the charge of the self-assembling peptide chain gradually decreases. When the self-assembling peptide is in electrostatic equilibrium, the intermolecular repulsion decreases, and weak interactions such as hydrophobic force, van der Waals force, and ionic bond drive the self-assembling peptide to self-assemble into a mesh scaffold structure, that is, the isoelectric point is around 6.2, and the self-assembling peptide solution forms a hydrogel on a macro scale. As the pH continues to rise, COO - Increase, NH3 + By removing a proton to form uncharged NH2, the self-assembled peptide chain becomes negatively charged as a whole and transforms from a gel peptide to a liquid state due to electrostatic interaction.

[0285] Experiment 7: Characterization of the self-assembling peptide gel of the present invention and cell culture on the gel

[0286] Experimental method: Taking SEQ ID NO: 24 as an example, the self-assembling peptide was dissolved in PBS, and a 2 wt % self-assembling peptide solution was prepared and sterilized for use as a self-assembling peptide mother solution.

[0287] Characterization of response layer structure: Take a certain amount of self-assembling peptide mother solution and dilute it to 0.5wt% with DMEM cell culture complete medium. Take 30μL of 0.5wt% self-assembling peptide solution and add it to a 96-well plate treated with tissue culture (TC). Leave it at room temperature overnight to form a gel. Tilt the culture plate and discard the supernatant. Put the 96-well plate into a freeze-drying machine precooled to minus 50℃ and freeze-dry it after precooling for 2h. The freeze-dried sample was sprayed with gold before imaging observation.

[0288] Cell 3D culture: dilute the self-assembling peptide stock solution to 1.0wt% with an equal volume of complete culture medium. Take 30μL of 0.5wt% self-assembling peptide solution and add it to a 96-well plate treated with tissue culture (TC). Place it in a 37℃ incubator overnight to form a gel. Tilt the culture plate and discard the supernatant. 5 100 μL of cell suspension with a concentration of 10 cells / mL was inoculated on the gel, and the culture medium was replaced every 2-3 days.

[0289] Experimental conclusion: After adding 0.5 wt% of the self-assembling peptide of the present invention to the culture wells, a stable gel layer was formed at the bottom of the culture wells after overnight incubation. Fig.17 It can be seen that after freeze-drying, the self-assembling peptide gel coating formed on the well plate has a regular porous three-dimensional structure, which can provide a sufficient three-dimensional microenvironment for cells and provide a good channel for gas-liquid exchange. It is possible that the close combination of high-concentration self-assembling peptide chains formed a stable self-assembling peptide coating.

[0290] The experiment of culturing cells on gel found that ( Fig.18 ), liver cancer cells (HepG2 cells) can be supported on the formed hydrogel layer and subsequently grow and proliferate in the form of cell spheres. As the culture time increases, the cell spheres become more and more compact and their diameters always increase at a similar rate, indicating that HepG2 cells can proliferate stably at a uniform rate during the 5-day culture. The relatively uniform proliferation rate of the cells also indicates that they are in good condition during the culture period and will not cause damage to the cells.

[0291] This experiment uses the TC-treated culture container as an example to prove that the self-assembling peptide of the present invention can react on the surface of the culture vessel to form a stable hydrogel, which can be used for 3D cell culture on the hydrogel; at the same time, this method is very easy to operate such as changing cell fluid, and the hydrogel is transparent and colorless, which is also convenient for imaging characterization.

[0292] Example 6: Application of the peptide hydrogel material of the present invention in the field of 3D cell culture

[0293] Experiment 1: The supporting effect of the hydrogel of the present invention on cells

[0294] Experimental method: Taking SEQ ID NO:9 as an example, HepG2 liver cancer cells were cultured in a cell culture flask in a mild and humid environment at 37°C and 5% CO2 using a high-glucose DMEM medium with a serum content of 10%. After culture, the cells were collected and resuspended in PBS buffer solution to obtain a concentration of 5×10 5 Cell suspension of cells / mL. The prepared cell suspension and Calcein-AM / PI dye solution were mixed evenly in a volume ratio of 2:1, and incubated in the dark at 37°C for 15 minutes. Then different volumes of 2wt% self-assembling peptide mother solution were added and mixed with the cell suspension after incubation in the dark, so that the total volume in the glass bottom culture dish was 200μL and the final concentration of the self-assembling peptide was 0.1wt%, 0.3wt%, and 0.5wt%. When preparing the control group samples, an equal volume of PBS buffer was used to replace the hydrogel material of the present invention in the experimental group. Subsequently, each experimental sample was observed using a LSM 980with Airyscan2 fast super-resolution laser confocal microscope.

[0295] Experimental conclusion: Fig.19 The results showed that in the control group without the hydrogel of the present invention, the cells naturally sank to the bottom of the glass-bottom culture dish without being supported, and were distributed in a 2D plane; when different concentrations of self-assembling peptides were introduced into the system, the cells in the system were supported by the three-dimensional network scaffold formed by the self-assembly of the self-assembling peptides, showing a three-dimensional distribution in a 3D environment.

[0296] This experimental phenomenon proves that the hydrogel material of the present invention, i.e., the three-dimensional network scaffold self-assembled by the self-assembling peptide, can effectively support cells, which is conducive to the growth and cultivation of cells in three-dimensional space in vitro, preventing them from sinking and adhering to the wall and then growing in a two-dimensional environment. Moreover, the entire initiation process is mild, no additional new substances need to be added, and the use is effective, convenient, and harmless to cells, showing the great potential of the self-assembling peptide material of the present invention in the field of cell culture and biomedicine.

[0297] The results of the experiments of the self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0298] Experiment 2: Application of 3D Cell Storage

[0299] Experimental method: Taking SEQ ID NO: 22 as an example, this experiment takes the storage of mouse mesenchymal stem cells at 4°C as an example. The separated mouse mesenchymal stem cells were evenly pipetted and 10 mL of equal amount was dispensed into cryopreservation tubes and pipetted evenly to make the cell concentration between 1×10 6The 3D group was prepared by adding mouse serum and 1% 50% MAP+50% SFEM storage solution of dual antibodies, and the self-assembling peptide SEQ ID NO: 22 was added to a final concentration of 0.05wt%; the 2D group was prepared by adding mouse serum and 1% 50% MAP+50% SFEM storage solution of dual antibodies, and then stored in a 4°C refrigerator.

[0300] Cell viability test:

[0301] (1) Mix the stored cells thoroughly by pipetting, take 500 μL of the cell suspension into each 1.5 mL EP tube, add PBS to wash, and centrifuge at 1500 rpm for 10 min;

[0302] (2) Discard the supernatant, resuspend with 150 μL of 1% PBA solution, then add 150 μL of live and dead cell staining working solution, mix thoroughly, incubate at 37°C for 15 min in the dark, and detect using flow cytometry.

[0303] Experimental results: In order to understand the changes in the survival rate of mesenchymal cells during the storage period, the Calcein-AM / PI live / dead double staining kit was used to double-stain them, and flow cytometry was used to detect them. Fig. 20 As shown, starting from the second day of storage, the survival rate of mouse mesenchymal stem cells in the 3D storage group was significantly higher than that in the 2D storage group, with a significant statistical difference (91% vs. 79%). And on the fifth day of storage, the cell survival rate was still high (81% vs. 43%), and the survival rate increased by nearly 50%. This shows that the storage effect of the mesenchymal stem cell storage system of the present invention is better than that of traditional 2D storage, and the cell survival rate is greatly improved.

[0304] The results of the experiments of the self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0305] Experiment 3: Application of 3D culture of liver cancer cells

[0306] Experimental method: The inherent property of adherent cells is that they are easy to aggregate and form spheres under conditions that prevent them from adhering to a certain plane, that is, they are easy to grow into cell spheres in a three-dimensional space. Cell spheres have concentration gradients of oxygen, nutrients, and metabolic waste, which can simulate the various characteristics of solid tissues. They are an important 3D physiological model for studying the occurrence of solid tumors and stem cell differentiation, and are widely used in the biomedical field. Moreover, cell spheres are simpler than other 3D physiological models. During the research process, imaging analysis can be achieved through common experimental means, such as optical, fluorescent, and confocal microscopy, which simplifies the experimental process in the research. In this experiment, the adherent cells HepG2 cultured in the self-assembling peptide culture system of the present invention triggered by high-sugar culture medium were used to verify whether cell spheres could be formed to verify that cell culture similar to that in vivo can be achieved in vitro.

[0307] Prepare the solution with a concentration of 1×10 6 The concentration of 1×10 cells / mL cell suspension was prepared by 5 Taking SEQ ID NO:9 as an example, the concentration obtained is 1×10 5 Cell suspensions of 10 cells / mL were inoculated into 96-well plates to which the self-assembling peptides of the present invention had been added (the final concentration of the self-assembling peptides in each well was 0.1wt%, 0.3wt%, 0.5wt%). During the culture period, the culture medium in the upper part of the well plate was removed and replaced with new culture medium every 3 days.

[0308] The diameters of 50 cell spheres in the culture system were observed and recorded under an inverted microscope after 3 days, 7 days, 11 days and 15 days of culture, and the cell sphere diameter data collected on these three days were averaged and plotted to detect whether the HepG2 cells cultured in the self-assembling peptide of the present invention can achieve long-term in vitro proliferation, and to photograph the characteristic cell spheres.

[0309] In the EDU fluorescent staining experiment, the liver cancer cells were cultured to an appropriate generation, the complete medium in the culture flask was aspirated, the cell surface was washed 1-2 times with PBS buffer, trypsin containing EDTA was added and incubated at 37°C for 1 min to allow the cells to be fully digested, complete medium was added to stop digestion, and the culture flask was rinsed with complete medium to remove the remaining cells, centrifuged at 800 rpm for 3 min, counted using a cell counting plate, and the cell suspension was diluted to 5×10 with complete medium. 4 cells / mL, seeded in a 48-well plate, the concentration of the self-assembling peptide was selected to be 0.3wt%, and stained with calcein-AM and EDU kits on the 3rd, 7th, 11th, and 15th days of culture, respectively, and photographed under a confocal fluorescence microscope.

[0310] Test results: Fig.21It can be seen that in the culture system containing 0.1wt.%, 0.3wt.%, 0.5wt.% of the triggered self-assembling peptide of the present invention, HepG2 cells are supported and subsequently grow and proliferate in the form of cell spheres. As the culture time increases, the cell spheres become more and more compact and their diameters always increase at a similar rate, indicating that HepG2 cells can proliferate stably at a uniform rate during the 15-day culture, that is, the culture system containing the triggered self-assembling peptide of the present invention can be used for long-term in vitro three-dimensional culture of cells. The relatively uniform proliferation rate of the cells also indicates that they are in good condition during the culture period and will not cause damage to the cells.

[0311] The bright field images show that the self-assembling peptide promotes cell aggregation. Therefore, the state of cell sphere culture and whether it proliferates are observed by confocal fluorescence microscopy. Fig. 22 A It can be found that the activity of the cell sphere is getting better and better. Most of the HepG2 spheroids cultured in the self-assembling peptide of the present invention are calcein AM positive, and there are few dead cells, which confirms the high cell viability and no necrotic core during the culture process. This can also prove that the self-assembling peptide of the present invention can simulate the extracellular matrix and provide a very suitable growth environment for the growth and survival of cells, thereby achieving the optimal effect of in vivo culture.

[0312] The proliferation of HepG2 spheroids cultured in hydrogels was further assessed by imaging with 5-ethynyl-2′-deoxyuridine (EDU). Fig. 22 B It can be found that the proliferating cells can be observed to be randomly distributed throughout the HepG2 sphere, indicating that sufficient nutrient diffusion occurs through the self-assembling peptide and sphere of the present invention during the culture process. As the number of days increases, it is found that the proliferation decreases on the 15th day, which indicates that the cells begin to approach differentiation as the proliferation decreases.

[0313] The results of the experiments of the self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0314] Experiment 4: Application of 3D culture of porcine muscle satellite cells

[0315] Experimental method: The culture of porcine muscle satellite cells is similar to the 3D culture of liver cancer cells in Experiment 3. Prepare a concentration of 1×10 6 The concentration of 1×10 cells / mL cell suspension was prepared by 5 Taking SEQ ID NO:21 as an example, the concentration obtained is 1×10 5Cells / mL of cell suspension were inoculated into 96-well plates to which the self-assembling peptide of the present invention had been added (the final concentration of the self-assembling peptide in each well was 0.3 wt.%). The plate culture medium was removed every 3 days during the culture period, and the cells were centrifuged and precipitated, and then resuspended in fresh culture medium containing 0.3 wt.% and continued to be cultured.

[0316] Experimental results: On the sixth day of culture, the diameters of 50 cell spheres in the culture system were observed and recorded under an inverted microscope to detect whether the porcine muscle satellite cells cultured in the self-assembling peptide of the present invention could achieve long-term in vitro proliferation, and the characteristic cell spheres were photographed ( Fig.23 ).Depend on Fig.23 It can be seen that in the culture system containing 0.3wt% of the self-assembling peptide of the present invention, the pig muscle satellite cells are supported and subsequently grow and proliferate in the form of cell balls. As the culture time increases, the cell balls become more and more compact, forming cell clusters with higher density and three-dimensional distribution, indicating that the pig muscle satellite cells proliferate well in a period of 6 days, that is, the self-assembling peptide system of the present invention forms a 3D support structure after initiation, which can be used for long-term in vitro three-dimensional culture of cells. At the same time, live-dead staining found that most of the cells were living cells, which were in good condition during the culture period and did not cause damage to the cells.

[0317] The experiments in Experiments 1-4 of this Example all prove that the culture system of the self-assembling peptide of the present invention has excellent cell support, and Experiment 2 also proves that the self-assembling peptide of the present invention has good biocompatibility and can be used for cell storage. Experiments 3 and 4 respectively prove that adding the self-assembling peptide of the present invention to the cell culture medium can highly restore the growth environment of cells in vivo and achieve long-term three-dimensional cell culture in vitro.

[0318] The results of the experiments of the self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0319] Experiment 5: Construction of organoids

[0320] Experimental method: This experiment takes SEQ ID NO:3 as an example to study the application of the hydrogel formed by the self-assembling polypeptide of the present invention in organoid construction. The received esophageal cancer tissues were placed in the treatment solution (5mL PBS + 10% double antibody solution) and soaked for 5 minutes; the tissues were cut into pieces, placed in the digestion solution, and placed in a shaker (37°C, 200r) for 1 hour. Prepare a 100μm cell sieve, filter the cell suspension with the cell sieve, resuspend in F12 medium, centrifuge the filtrate, 250g, 5min, and discard the supernatant. Add 5mL of F12 medium to the esophageal cell and paracancerous cell pellets, centrifuge again, 250g, 5min, and discard the supernatant. Mix the precipitated cells with 1wt% of the polypeptide (SEQ ID NO:3) self-assembled hydrogel solution to make the final cell concentration of 4×10 6 / mL. Take 200μL of the cell-hydrogel mixture, gently blow and mix, then add three drops per well on a low-adhesion 24-well cell culture plate, add three drops in total, then put the cell culture plate in an incubator upside down overnight, take out the cell culture plate the next day, add 500μl of special culture medium per well and put it in an incubator for culture. Observe every day and change the medium every two days.

[0321] Experimental results: After 7 days of continuous culture, esophageal primary cells can proliferate into spherical structures with organoid morphology ( Fig.24 ). This experiment proves that for the primary tumor cells of the patient, the hydrogel formed by the polypeptide of the present invention can stably support the tumor cells to form a 3D distribution, and then under the action of the organoid-specific culture medium, a functional structure with a preliminary organ morphology is formed, which can be used for drug screening, animal model construction and other applications. The results of this experiment can be extended to the culture application of stem cell differentiation organoids such as induced pluripotent stem cells and embryonic stem cells, and the culture application of organoids of other primary cells except tumors.

[0322] The results of the experiments of the self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0323] Example 7: Application of the self-assembling peptide of the present invention as a microsphere dispersant

[0324] Experiment 1: Verification of the support of self-assembled network scaffolds for polylactic acid microspheres and polystyrene microspheres

[0325] Experimental method: The self-assembling peptide of the present invention self-assembles to generate a network scaffold that has good dispersion support functions not only for cells, but also for nano- and micron-sized microspheres and microcarriers, drugs, and functional molecules. This study takes SEQ ID NO:23 as an example to study the support function of the network scaffold for polylactic acid and polystyrene microspheres, and at the same time verify the effect of the microsphere / self-assembling peptide composite system on cell proliferation. 1wt% of the self-assembling peptide mother solution and microsphere dry powder were added to the culture medium respectively, until the final concentration of the self-assembling peptide was 0.1wt% and the final concentration of the microspheres was 1wt%; at the same time, 1wt% of the microspheres were added to the basal culture medium as a control to study the dispersion effect of the self-assembling peptide on the microspheres. The microspheres used in this experiment were prepared in the laboratory, with a particle size between 150-500μm.

[0326] Experimental results: Fig.25 As shown in AB, the left picture is a microsphere solution without self-assembling peptides. The polylactic acid microspheres (A) and polystyrene microspheres (B) all sink to the bottom of the bottle after 30 minutes. After adding 1wt% of self-assembling peptides (right picture), the microspheres can be evenly distributed in 3D in the system. The obtained self-assembling peptide mixed solution containing microspheres was placed at room temperature for 3 months, and it can be seen that the obtained solutions are all in a uniform suspension state, and there is no obvious sedimentation and separation phenomenon.

[0327] This experimental phenomenon proves that the three-dimensional network scaffold formed by the self-assembly peptide of the present invention can effectively support microspheres other than cells, and the microspheres play a good role in dispersion and support in the solution. The whole process of the experiment does not require the addition of other ingredients, and the operation is convenient. The large-size microspheres used in this experiment fully verify the support ability of the self-assembly peptide of the present invention to form a network scaffold.

[0328] Experiment 2: Application of self-assembled network scaffold / microcarrier composite system for cell proliferation

[0329] Experimental method: Large-size microspheres can be used as microcarriers for cell culture; this study continued to use SEQ ID NO: 23 as an example to verify the effect of the microsphere / self-assembling peptide composite system on cell proliferation. In the cell culture experiment, the sterile microspheres were first added to a 48-well plate at a final concentration of 1wt%, and then the cultured porcine muscle satellite cells were inoculated at 5×10 4 1 cell was added to each well of the culture plate, and the cells were first incubated with the microspheres for 1 day, and then a 2 wt % self-assembling peptide mother solution was added to a final concentration of 0.1 wt % for culture. The cells were counted on the 1st, 3rd, 5th and 7th days. The culture medium in the wells was first aspirated, and PBS was added for washing 2-3 times. Then, trypsin containing EDTA was added for digestion for about 1 hour to make the cells fall off the carrier, and then the cells were counted using a cell counting plate.

[0330] The CCK8 method was used to determine the cell viability of different groups and evaluate their cytotoxicity. As described above, the cells on the 1st, 3rd, 5th, and 7th days of culture were measured, and the cell morphology was observed by confocal microscopy, using Calcein-AM / PI staining solution.

[0331] Experimental results: Porcine muscle satellite cells were selected for the following biological experiments. The effects of the dual system of microcarriers + self-assembling peptides on cell proliferation and activity were detected by using cell counting and CCK8. A 2D group, a microcarrier group, and a microcarrier + self-assembling peptide group were set up. Cell counting and activity experiments were performed on the 1st, 3rd, 5th, and 7th days, respectively. The microsphere group was in a static culture state without using a bioreactor. The concentration of the self-assembling peptide was 0.1wt%. Since Calcein-AM / PI dye can stain fire cells, the fluorescence brightness reflects the cell density. The results are as follows Fig.26 AD and Fig. 27Figures AD show the changes in cell density on microspheres during proliferation of polylactic acid microspheres / self-assembling peptide composite system and polystyrene microspheres / self-assembling peptide composite system. It can be seen that with the extension of culture time, the cell density on polylactic acid microspheres and polystyrene microspheres increased significantly; both composite systems showed that only a small number of cells attached on the first day, gradually became elliptical and flattened within 1 day, and on the third day, cells began to stretch and adhere to the surface of the microspheres. On the 7th day, the cells had proliferated in large numbers and wrapped around the entire surface of the microspheres.

[0332] Through the characterization of the proliferation effect and cell viability of pig muscle satellite cells, it was found that Fig.28 (polylactic acid / self-assembling peptide) and Fig.29 By comparing the cell growth density and cell activity curve in (polystyrene microspheres / self-assembling peptides), the microsphere / self-assembling peptide composite system showed better results. This experiment can further prove that after the added self-assembling peptides support the microcarriers, the contact area between the microspheres and cells is increased, which can accelerate the circulation of nutrients and air, which greatly promotes cell proliferation and changes in cell status.

[0333] The results of the experiments of the self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0334] Example 8: Research and application of cell migration (transwell)

[0335] Experimental method: This experiment takes SEQ ID NO:24 as an example. Dissolve the polypeptide lyophilized powder in serum-free medium to prepare a 10 mg / mL mother solution, and place it at 4°C for use; dilute the polypeptide mother solution to a concentration of 1 mg / mL with serum-free medium; draw 80 μL of the diluted polypeptide solution and add it to the bottom of the chamber along the wall of the chamber (the glue liquid surface is horizontal and the thickness of the glue is uniform) Place the chamber in the culture plate and incubate at 37°C for 4 hours to solidify the glue into a thin film; take out the chamber, suck off the excess liquid on the surface of the chamber, add 100 μL of serum-free medium, and incubate in a 37°C incubator for 30 minutes to gel; take out the chamber, suck off the liquid in the chamber, and check whether there is liquid passing through the chamber into the lower chamber. If not, cells can be inoculated; starve the cells for 12 hours in advance, digest the cells, and dilute them with serum-free medium until the cells are 7×10 5 / mL. Prepare a medium containing 20% ​​serum and add it to a 24-well plate, 600μL per well. Use sterilized tweezers to gently place the chamber into the well plate (avoid bubbles); after mixing the cell suspension, take 200μL and gently add it to the chamber, cover the well plate, and culture in a 37℃ incubator for 48h.

[0336] Experimental results: Fig.30(Photo taken under a 4X microscope) As shown, polypeptide hydrogels were used to simulate the typical matrix encountered by HepG2 cells (human liver cancer cells) and Hela cells during invasion in vivo. The crystal violet staining results showed that both HepG2 and Hela cells would degrade the adjacent matrix and migrate through the ECM gel to complete the invasion, thereby adhering to the bottom of the culture well plate and proliferating.

[0337] Example 9: Application of tumor transplantation in animals

[0338] Experiment 1: Orthotopic liver tumor implantation in mice

[0339] Experimental method: This experiment uses SEQ ID NO: 22, and takes the liver orthotopic tumor of mice as an example to verify the application of the patent of this invention as a tissue engineering material in the construction of animal models. C57BL / 6 mice were randomly divided into a control group and an experimental group, with 10 mice in each group. The mice were anesthetized by intraperitoneal injection of 0.6% sodium pentobarbital, fixed on a mouse board in a supine position, and routinely disinfected with 75% ethanol. The abdominal skin was incised transversely under the xiphoid process, and the chest cavity was gently pushed to expose the left lateral lobe of the liver to the surface. The microinjector was inserted into the liver about 1 cm, and 30 μL containing 2×10 6 EGFP-Luc-Hepa1-6 cells (mouse hepatoma cells) suspension in PBS (control group) or 30 μL containing 2×10 6 The self-assembled polypeptide (final polypeptide concentration is 0.75wt%) suspension of EGFP-Luc-Hepa1-6 cells was placed in the experimental group. The microinjector was slowly pulled out and the pinhole was sealed with medical glue. The abdomen was sutured layer by layer and disinfected with ethanol again. Mice were intraperitoneally injected with 15mg / mL D-luciferin, anesthetized with isoflurane oxygen inhalation, and placed in the supine position in the dark box of the LuminaⅡ imaging system for in situ live imaging of liver cancer.

[0340] Experimental results: After 18 days of culture, the tumor-grafted livers of the mice in the experimental and control groups were dissected as follows: Fig.31 As shown in the figure, the results of the control group (B) showed that the liver tumor had an irregular morphology, nodular infiltration growth, uneven surface, and hard texture. The results of the experimental group (A) showed that after being wrapped with polypeptide hydrogel, the inoculated cells formed a tumor mass into a whole mass, which was convenient for statistical analysis; while the tumor cell mass in the group without polypeptide was scattered in other parts of the liver, which was not conducive to statistical observation and comparison of results.

[0341] Experiment 2: Subcutaneous tumor implantation in mice

[0342] Experimental method: This experiment uses SEQ ID NO:7 and takes mouse subcutaneous tumor implantation as an example to verify the application of the present invention as a tissue engineering material in the construction of animal models. SCID Beige mice were divided into a control group and an experimental group, with 5 mice in each group. The mice were anesthetized by intraperitoneal injection of 0.6% sodium pentobarbital, fixed on a mouse board in a supine position, and routinely disinfected with 75% ethanol. The abdominal skin was cut horizontally, and the microinjector was inserted into the subcutaneous part of the abdomen, and 30 μL containing 3×10 6 N87 cells (human gastric cancer cells) in PBS (control group) or 30 μL of PBS containing 3×10 6 The self-assembled polypeptide of N87 cells (the final concentration of polypeptide is 0.75wt%) was placed in the gel suspension (experimental group). The microinjector was slowly pulled out and the needle hole was sealed with medical glue. The abdomen was sutured layer by layer and disinfected with ethanol again. The two groups of mice were cultured for 20 consecutive days.

[0343] Experimental results: Comparison found that ( Fig.32 AB), the subcutaneous tumor implantation of the experimental group mice was significantly faster than that of the control group. On the 18th day, the diameter of the tumor implanted in the experimental group was 30% larger than that of the control group. The tumor implanted in the experimental group was regular in shape, while the tumor implanted in the control group was irregular in shape and smaller in size. This experiment once again proves the application of the hydrogel formed by the self-assembling polypeptide of the present invention as an embedding material for cells or functional components in animal models.

[0344] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A self-assembling peptide comprising a hydrophilic domain and an RGD domain, wherein the hydrophilic domain comprises at least one β-turn region capable of forming a β-turn, and the RGD domain comprises an amino acid sequence of arginine (R)-glycine (G)-aspartic acid (D).

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

3. The self-assembling peptide according to claim 1, wherein the β-turn region comprises a β-turn motif formed by 3-6 amino acids, and the β-turn motif has the following structure: X1X2X3, X1X2X3X4, X1X2X3X4X5, or X1X2X3X4X5X6, in, X1, X2, X3, X4, X5, and X6 are amino acid residues. In each β-turn motif, X1, X2, X3, X4, X5, and X6 are the same as or different from each other.

4. The self-assembling peptide according to claim 3, comprising one hydroxyproline (O), preferably said hydroxyproline (O) is contained in said β-turn motif. The self-assembling peptide according to claim 1 , wherein the hydrophilic domain comprises 1 to 8 β-turn regions.

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

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

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

9. The self-assembling peptide according to claim 3, wherein the β-turn motif comprises an amino acid sequence selected from the group consisting of: GPGGE (SEQ ID NO:66), GOGAE (SEQ ID NO:67), GOGGAE (SEQ ID NO:68), GOGE (SEQ ID NO:69), GOGGE (SEQ ID NO:70), GPGAD (SEQ ID NO:71), GOGGD (SEQ ID NO:72), GPGGV (SEQ ID NO:73), GOGGV (SEQ ID NO:74), GPGGK (SEQ ID NO:75), GOGGK (SEQ ID NO:76), GPGAE (SEQ ID NO:77), GOGAD (SEQ ID NO:78), GPAAD (SEQ ID NO:79), GOAAE (SEQ ID NO:80), GPGGD (SEQ ID NO:81), GPGGGV (SEQ ID NO:82), GPGV (SEQ ID NO:83), OGGAE (SEQ ID NO:84) or GOGVI (SEQ ID NO:88).

10. The self-assembling peptide according to claim 1, wherein the RGD domain comprises or is linked to one or more amino acids selected from glycine (G), proline (P) or hydroxyproline (O), Preferably, the RGD domain comprises or is linked to an amino acid selected from glycine (G)-proline (P) or glycine (G)-hydroxyproline (O).

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

12. The self-assembling peptide according to claim 1, wherein the hydrophobic domain comprises an amino acid sequence selected from the group consisting of: FIIII (SEQ ID NO:32), IIII (SEQ ID NO:33), IIII (SEQ ID NO:34), ILILI (SEQ ID NO:35), FLFLF (SEQ ID NO:36), IVIVI (SEQ ID NO:37), VLFIIV (SEQ ID NO:38), VLIII (SEQ ID NO:39), IVALF (SEQ ID NO:39) NO:40), LFIVL (SEQ ID NO:41), FIAIV (SEQ ID NO:42), FIIIV (SEQ ID NO:43), Ac-VLFIIV (SEQ ID NO:44), Ac-IVIVI (SEQ ID NO:45), Ac-IIIIII (SEQ ID NO:46), IIIIII (SEQ ID NO:47), FLIVI (SEQ ID NO:48), FLIIA (SEQ ID NO:49), FIFIF (SEQ ID NO:50), IFIFI (SEQ ID NO:51), IAILI (SEQ ID NO:52), LLLLL (SEQ ID NO:53) or VIVIV (SEQ ID NO:54).

13. The self-assembling peptide according to claim 11, wherein the RGD domain is located at the end of the self-assembling peptide, preferably at the end of the self-assembling peptide close to the hydrophilic domain, or The RGD domain is located between the hydrophobic domain and the hydrophilic domain.

14. The self-assembling peptide according to claim 1, further comprising a connecting domain, Preferably, the linking domain comprises 2-8 amino acid residues, preferably 4-5 amino acid residues, Preferably, the linker domain comprises small side chain amino acids, amino acids with hydroxyl groups on the side chains and / or hydrophobic amino acids far from the hydrophobic region. Preferably, the small side chain amino acid is selected from glycine (G), alanine (A) and serine (S), the amino acid with a hydroxyl group on the side chain is selected from serine (S), threonine (T) and hydroxyproline (O), the hydrophobic amino acid away from the hydrophobic domain is selected from I, V, L, F and A, and the hydrophobic amino acids I, V, F, L, A are interchangeable. Preferably, the linking domain comprises an amino acid sequence selected from the group consisting of: GSII, GSGII, GSVI, GOII, OGII, or GTVI, where: S, T, and O can be interchanged. More preferably, the connecting domain comprises an amino acid sequence selected from the group consisting of: GSII (SEQ ID NO:55), GTII (SEQ ID NO:56), GTVI (SEQ ID NO:57), GOVI (SEQ ID NO:58), GSVI (SEQ ID NO:59), GSGII (SEQ ID NO:60), GSGVI (SEQ ID NO:61), GOII (SEQ ID NO:62), OGII (SEQ ID NO:63), GOGVI (SEQ ID NO:63) ID NO:64) or GOGII (SEQ ID NO:65).

15. The self-assembling peptide according to claim 14, wherein the connecting domain is located between the hydrophobic domain and the hydrophilic domain, or The connecting domain is located between the hydrophobic domain and the RGD domain, or The linker domain is located between the hydrophilic domain and the RGD domain.

16. The self-assembling peptide according to claim 1, comprising at least one amino acid sequence selected from the group consisting of the following SEQ ID NOs: 1-4 and SEQ ID NOs: 6-29.

17. A method for preparing the self-assembling peptide according to any one of claims 1 to 16, characterized in that: The polypeptide is prepared by a method selected from solid phase polypeptide synthesis, microbial synthesis or fermentation.

18. A method for forming a three-dimensional network material from the self-assembling peptide according to any one of claims 1 to 16, the method comprising the step of inducing the self-assembling peptide to form a three-dimensional network material under a condition of pH less than 9.

19. The preparation method according to claim 18, comprising initiating the self-assembling peptide solution at a pH less than 9, preferably at a pH of 6-7.5, Preferably, in the self-assembling peptide solution, the concentration of the self-assembling peptide is ≤ 0.09wt%, preferably 0.0001wt%-0.09wt%, more preferably 0.01wt%-0.09wt%, and the initiation is performed to obtain a three-dimensional network material in a solution state; Preferably, in the self-assembling peptide solution, the concentration of the self-assembling peptide is ≥ 0.1 wt%, preferably > 0.3 wt%, and more preferably 0.3 wt%-4 wt%, and the initiation is performed to obtain a three-dimensional network material in a hydrogel state.

20. The preparation method according to claim 18, further comprising: The self-assembling peptide is completely dissolved under alkaline conditions, preferably under conditions with a pH value greater than 7.4, and more preferably in an aqueous solvent with a pH value greater than 9.

21. A three-dimensional network material comprising the self-assembling peptide according to any one of claims 1 to 16, or prepared by the method according to any one of claims 18 to 20.

22. The three-dimensional network material according to claim 21, wherein the three-dimensional network material is in a solution state, and preferably, in the three-dimensional network material, the concentration of the self-assembling peptide is ≤ 0.1wt%; more preferably 0.0001wt%-0.09wt%; further preferably 0.01wt%-0.09wt%; or The three-dimensional network material is in a hydrogel state. Preferably, in the three-dimensional network material, the concentration of the self-assembling peptide is ≥ 0.1 wt%, preferably > 0.3 wt%, and more preferably 0.3 wt%-4 wt%; or The three-dimensional network material is in a dry powder state, and preferably, is obtained from the three-dimensional network material in a hydrogel state by freeze-drying, spray-drying, or drying.

23. A composite system comprising the self-assembling peptide according to any one of claims 1 to 16 and a microcarrier.

24. The composite system according to claim 23, wherein the microcarrier comprises at least one of a polystyrene microcarrier, a polylactic acid microcarrier, a macroporous gelatin microcarrier, a methacrylate gelatin microcarrier, a laminarin microcarrier, a carboxybetaine microcarrier, an isopropylacrylamide microcarrier, a polyvinyl alcohol / cellulose microcarrier, a D,L-lactic acid-co-ethylene glycol microcarrier, a D,L-lactic acid-co-ethylene glycol microcarrier, a D,L-lactic acid-co-ethylene glycol microcarrier, a D,L-lactic acid-co-ethylene glycol / chitosan microcarrier, a collagen microcarrier, a chitosan microcarrier, a chitin microcarrier, a PHEMA microcarrier, a polyurethane foam microcarrier, a polylactide microcarrier, an alginate gel microcarrier and a magnetic microcarrier; Preferably, the particle size of the microcarrier is 50-500 μm, preferably 150-500 μm.

25. According to the composite system of claim 23, the self-assembling peptide and the microcarrier are placed in the same container in the form of powder; or, the self-assembling peptide and the microcarrier are placed in different containers in the form of powder; or, the self-assembling peptide is placed in one container in the form of solution, and the microcarrier is placed in another container in the form of powder; or, the self-assembling peptide is placed in one container in the form of powder, and the microcarrier is placed in another container in the form of solution; or the self-assembling peptide and the microcarrier are placed in the same container in the form of solution.

26. A composition comprising the self-assembling peptide according to any one of claims 1 to 16, the composite system according to any one of claims 23 to 25, and the aqueous solvent and / or the acidic solution for adjusting pH according to claim 20.

27. Use of the self-assembling peptide of any one of claims 1-16, the method of any one of claims 17-20, the three-dimensional network material of claim 21 or 22, the composite system of any one of claims 23-25 ​​or the composition of claim 26 in one or more selected from the following: in vitro three-dimensional storage and culture of cells, cell migration test, organoid construction, in vitro three-dimensional storage and culture of viruses, tissue engineering scaffolds or tissue filling materials, microsphere and microcarrier dispersions or embedding fillings, drug, vaccine and compound storage and application carriers, medical dressings or medical cosmetic materials, tissue engineering scaffolds, stem cell transplantation, tumor animal transplantation, gene therapy, stem cell therapy, tissue repair, medical cosmetology, etc.

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