Self-assembled peptide and use thereof

Abstract

Provided are a self-assembled peptide, a three-dimensional network material formed by the self-assembled peptide, and the use of the three-dimensional network material. The self-assembled peptide 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). The self-assembled peptide can self-assemble to form a three-dimensional network material in a neutral solution without initiation by any other exogenous components.

Description

Self-assembling peptides and their applications Technical Field

[0001] The present 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 considerable attention due to their excellent biocompatibility and diverse functionality. Peptides are composed of amino acids linked by amide bonds. Due to the diverse chemical structures of amino acid residues, peptides can effectively self-assemble through hydrogen bonding between peptide bonds, hydrogen bonding between amino acid residues, electrostatic interactions, hydrophobic interactions, and π-π stacking interactions, thereby spontaneously forming structured aggregates 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 increasing attention in the fields of stem cells, tissue engineering and even the entire biomedicine.

[0004] Most polypeptide self-assembly 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 support and repair cells, functional microspheres, functional molecules, and drugs in both solution and hydrogel states, and to develop their applications in the fields of cell 3D culture, medical aesthetic regeneration and repair, and implant carriers.

[0006] Summary of the Invention

[0007] The invention provides a self-assembling peptide containing an RGD cell adhesion sequence, which can self-assemble into a three-dimensional network material in a neutral solution without any other exogenous components being needed to initiate the self-assembly.

[0008] 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).

[0009] 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 a terminal end, preferably comprises or is linked to one acidic amino acid or hydrophobic amino acid.

[0010] In some embodiments, at least one β-turn region comprises an acidic amino acid or a hydrophobic amino acid at a terminus.

[0011] 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).

[0012] 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:

[0013] X1X2X3, X1X2X3X4, X1X2X3X4X5, or X1X2X3X4X5X6,

[0014] Wherein, X1, X2, X3, X4, X5, and X6 are amino acid residues, and X1, X2, X3, X4, X5, and X6 in each β-turn motif are identical to or different from each other.

[0015] In some embodiments, the self-assembling peptide comprises one hydroxyproline (O).

[0016] In some embodiments, the β-turn motif comprises one hydroxyproline (O), and preferably, X2 is hydroxyproline (O).

[0017] In some embodiments, the hydrophilic domain comprises 1-8 β-turn regions.

[0018] In some embodiments, the hydrophilic domain comprises 1, 2, 3, 4, 5, 6, 7, or 8 β-turn regions.

[0019] 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.

[0020] In some embodiments, the β-turn motif in the β-turn region comprises or is linked to an acidic amino acid.

[0021] 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).

[0022] In some embodiments, the hydrophilic domain comprises two β-turn regions, and the ends of the β-turn regions comprise acidic amino acids E.

[0023] 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.

[0024] In some embodiments, the hydrophilic domain comprises two β-turn regions, and the ends of the β-turn regions comprise acidic amino acids D.

[0025] In some embodiments, the hydrophilic domain comprises two β-turn regions, wherein one β-turn region comprises an acidic amino acid D at its end, and the other β-turn region comprises an amino acid V at its end.

[0026] 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.

[0027] In some embodiments, the hydrophilic domain comprises two β-turn regions, wherein one end of the β-turn region comprises the acidic amino acid E, and the other end of the β-turn region comprises the amino acid V.

[0028] 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.

[0029] In some embodiments, the hydrophilic domain comprises a β-turn region, wherein a terminus of the β-turn region comprises amino acid V.

[0030] 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.

[0031] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X2 is O.

[0032] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X1 is G.

[0033] 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.

[0034] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X2 is P.

[0035] In some embodiments, the hydrophilic domain comprises two β-turn motifs in which X2 is P.

[0036] In some embodiments, one or more of X1, X3 and X4 is glycine (G).

[0037] In some embodiments, the β-turn motif comprises an amino acid sequence selected from the group consisting of:

[0038] GPGGE (SEQ ID NO:66), GOGAE (SEQ ID NO:67), GOGGAE (SEQ ID NO:68),

[0039] GOGE(SEQ ID NO:69), GOGGE(SEQ ID NO:70), GPGAD(SEQ ID NO:71),

[0040] GOGGD (SEQ ID NO:72), GPGGV (SEQ ID NO:73), GOGGV (SEQ ID NO:74),

[0041] GPGGK (SEQ ID NO:75), GOGGK (SEQ ID NO:76), GPGAE (SEQ ID NO:77),

[0042] GOGAD (SEQ ID NO:78), GPAAD (SEQ ID NO:79), GOAAE (SEQ ID NO:80),

[0043] GPGGD (SEQ ID NO:81), GPGGGV (SEQ ID NO:82), GPGV (SEQ ID NO:83),

[0044] OGGAE (SEQ ID NO: 84) or GOGVI (SEQ ID NO: 88).

[0045] In some embodiments, X1 and X4 form a hydrogen bond.

[0046] 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.

[0047] At least one beta-turn motif in the hydrophilic domain includes an alanine, thereby improving the mechanical properties of the self-assembling peptide.

[0048] In some embodiments, the N-terminus of the hydrophilic domain can be modified with an agent or group selected from the group consisting of carboxylic acid, thiol, ketoate, nitrite, phosphonate, thiophosphate, carbonate, sulfate, nitrate, vinyl sulfone, amide, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and / or silane modifications.

[0049] 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).

[0050] In some embodiments, the RGD domain comprises or is linked to an amino acid selected from the group consisting of glycine (G) and proline (P). In some embodiments, the RGD domain comprises or is linked to an amino acid selected from the group consisting of glycine (G) and hydroxyproline (O).

[0051] In some embodiments, the self-assembling peptide further comprises a hydrophobic domain, wherein the amino acid sequence of the hydrophilic domain is more hydrophilic than the amino acid sequence of the hydrophobic domain.

[0052] 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.

[0053] Preferably, the hydrophobic amino acid is selected from one or more of isoleucine (I), valine (V), leucine (L), phenylalanine (F) and alanine (A).

[0054] In some embodiments, the hydrophobic amino acid is selected from one or more of I, V, L, and F.

[0055] In some embodiments, the C-terminus of the hydrophobic domain is modified with an agent or group selected from the group consisting of acetyl, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and / or silane modifications.

[0056] In some embodiments, the hydrophobic domain comprises an amino acid sequence selected from the group consisting of:

[0057] FIIII (SEQ ID NO:32), IIII (SEQ ID NO:33), IIII (SEQ ID NO:34), ILILI (SEQ ID NO:35),

[0058] FLFLF (SEQ ID NO:36), IVIVI (SEQ ID NO:37), VLFIIV (SEQ ID NO:38),

[0059] VLIII (SEQ ID NO:39), IVALF (SEQ ID NO:40), LFIVL (SEQ ID NO:41),

[0060] FIAIV (SEQ ID NO:42), FIIIV (SEQ ID NO:43), Ac-VLFIIV (SEQ ID NO:44),

[0061] Ac-IVIVI (SEQ ID NO:45), Ac-IIIIII (SEQ ID NO:46), IIIIII (SEQ ID NO:47),

[0062] FLIVI(SEQ ID NO:48), FLIIA(SEQ ID NO:49), FIFIF(SEQ ID NO:50),

[0063] IFIFI (SEQ ID NO:51), IAILI (SEQ ID NO:52), LLLLL (SEQ ID NO:53) or VIVIV (SEQ ID NO:54).

[0064] 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.

[0065] In some embodiments, the RGD domain is located between the hydrophobic domain and the hydrophilic domain.

[0066] The amino acid sequence of the hydrophobic domain is hydrophobic compared to the amino acid sequence of the hydrophilic domain.

[0067] 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.

[0068] In some embodiments, the linker domain comprises 2-8 amino acid residues, preferably 4-5 amino acid residues.

[0069] In some embodiments, the linker domain comprises amino acids with small side chains, amino acids with hydroxyl groups on their side chains, and / or hydrophobic amino acids that are distal to the hydrophobic region.

[0070] In some embodiments, the amino acid with a smaller side chain is selected from glycine (G), alanine (A), and serine (S).

[0071] In some embodiments, the amino acid with a hydroxyl group on the side chain is selected from serine (S), threonine (T) and hydroxyproline (O),

[0072] In some embodiments, the hydrophobic amino acids away from the hydrophobic domain are selected from I, V, L, F and A, and the hydrophobic amino acids I, V, F, L, and A are interchangeable.

[0073] 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.

[0074] More preferably, the connecting domain comprises an amino acid sequence selected from the group consisting of:

[0075] 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).

[0076] 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.

[0077] 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.

[0078] In some embodiments, the self-assembling peptide has a length of 15-50 amino acids, preferably 15-25 amino acids.

[0079] 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.

[0080] 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:

[0081] The self-assembling peptide of the present invention having the above structure can be triggered to form a three-dimensional network material.

[0082] The three-dimensional network material has a nanostructure.

[0083] 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 polypeptide synthesis, microbial synthesis, fermentation or other biochemical methods.

[0084] 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 the 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 support and repair functions in a solution state.

[0085] Therefore, in a third aspect, the present invention provides a method for forming a three-dimensional network structure by initiating the self-assembling peptide of the first aspect, the method comprising the step of initiating the self-assembling peptide to form a three-dimensional network material under conditions of pH less than 9.

[0086] In some embodiments, the method comprises initiating the self-assembling peptide solution at a pH of 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 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 by itself without undergoing an obvious triggered assembly response to metal ions and proteins. For example, it can self-assemble in response to calcium ions, but has no significant response to magnesium ions and BSA.

[0087] In some embodiments, the self-assembling peptide solution is initiated at a pH of 6 to 9, 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.

[0088] 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.).

[0089] In some embodiments, the concentration of the self-assembling peptide in the self-assembling peptide solution is ≤0.09 wt%, preferably 0.0001 wt%-0.09 wt%, more preferably 0.01 wt%-0.09 wt%, and the initiation is performed to obtain a three-dimensional network material in a solution state.

[0090] In some embodiments, the concentration of the self-assembling peptide in the self-assembling peptide solution is ≥0.1wt%, preferably >0.3wt%, further preferably 0.3wt%-4wt%, and even more preferably 0.5wt%-1wt%, and the initiation is performed to obtain a three-dimensional network material in a hydrogel state.

[0091] In some embodiments, the self-assembling peptide solution can form a hydrogel membrane layer.

[0092] In some embodiments, the preparation method further comprises: completely dissolving the self-assembling peptide in an aqueous solvent under alkaline conditions, preferably at a pH greater than 7.4, more preferably at a pH greater than 9, to obtain a self-assembling peptide solution.

[0093] 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.

[0094] The solvent can be used to dissolve the self-assembling peptide of the present invention as long as it can provide an alkaline environment solution, preferably a physiologically acceptable solution.

[0095] 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, and adjusting the pH 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.

[0096] 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.

[0097] 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 %.

[0098] The present invention unexpectedly discovered that the self-assembling peptides can form a mesh scaffold that can be deposited to form a hydrogel film layer, and the resulting three-dimensional mesh scaffold structure is more stable. Therefore, such self-assembling peptides are particularly suitable for the application scenario of culturing cells on the hydrogel after coating in 3D cell culture. The three-dimensional network material is a nanostructure.

[0099] Furthermore, the hydrogel formed by the self-assembling peptides of the present invention at high concentrations exhibits self-repair capabilities. The hydrogel can be disrupted by mechanical force and then restored to its gel state after the force is removed. This recovery time does not exceed 10 minutes, and the storage modulus of the hydrogel after recovery is at least 70% of its pre-destruction value, preferably at least 85%, and more preferably at least 95% of its pre-destruction value.

[0100] 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 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.

[0101] It should be noted that if the hydrophobic domain is too hydrophobic, it is easy to form an association, and if the hydrophobic domain is too hydrophobic, 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 to not 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.

[0102] The present invention unexpectedly discovered that self-assembling peptides with the structures described herein can be triggered to self-assemble into nanonetwork structures under physiological conditions, in the presence of a wide range of endogenous substances in cell culture environments and the human body. In particular, the use of endogenous physiological substances, such as serum, plasma, cell culture media, plant and animal tissue fluids, and animal tissues, as initiators significantly reduces safety risks in clinical applications, which is particularly important for drug delivery, tissue repair, and regenerative medicine.

[0103] In a fourth aspect, the present invention provides a three-dimensional network material, comprising the self-assembling peptide of the first aspect.

[0104] In some embodiments, the three-dimensional network material is obtained using the method of the third aspect.

[0105] In some embodiments, the three-dimensional network material is in a solution state. Preferably, the concentration of the self-assembling peptide in the three-dimensional network material is ≤ 0.1 wt %, more preferably 0.0001 wt % to 0.09 wt %, and even more preferably 0.01 wt % to 0.09 wt %.

[0106] In some embodiments, the three-dimensional network material is in a hydrogel state. Preferably, the concentration of the self-assembling peptide in the three-dimensional network material is ≥0.1 wt%, preferably >0.3 wt%, and more preferably 0.3 wt%-4 wt%.

[0107] 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.

[0108] 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 freeze-dried powder of a hydrogel.

[0109] In some embodiments, the three-dimensional network material is in the form of an injectable hydrogel.

[0110] In some embodiments, the three-dimensional network material is a nanostructure.

[0111] The three-dimensional network material has more β-sheet structures than the self-assembling peptide.

[0112] In a fifth aspect, the present invention provides a composite system comprising the self-assembling peptide of the first aspect and a microcarrier.

[0113] In some embodiments, the microcarrier is a microsphere or a microslide.

[0114] In some embodiments, the microcarriers include at least one of polystyrene microcarriers, polylactic acid microcarriers, macroporous gelatin microcarriers, methacrylated 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.

[0115] In some embodiments, the particle size of the microcarrier is 50-500 μm, preferably 150-500 μm.

[0116] 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.

[0117] 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 self-assembled by self-assembling peptides, wherein the microcarriers are supported by the three-dimensional network material.

[0118] 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.

[0119] In a seventh aspect, the present invention further provides a method for culturing cells, comprising the step of culturing cells using the composite system of the fourth aspect or the cell culture system of the fifth aspect.

[0120] Before cell culture, the self-assembling peptide is prepared into a solution and then added to 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.

[0121] 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 concentrations. A higher three-dimensional network material concentration can support more microcarriers.

[0122] In the cell culture method of the present invention, cells are attached to microcarriers, which are supported by a three-dimensional network material formed by self-assembly of self-assembling peptides in the system. Although it is a static culture, similar to dynamic culture, the microcarriers are suspended in the system, evenly distributed and remain static.

[0123] The cell culture system of the present invention can provide a novel microcarrier culture cell pattern, that is, microcarriers are supported by three-dimensional network materials, and the uniform dispersion and suspension state of microcarriers are achieved, thereby better helping cells to adhere and promoting cell proliferation, and the microcarriers are always kept in a state of uniform dispersion in suspension throughout the cell culture cycle, without aggregation or precipitation. This pattern combines the advantages of static culture and dynamic culture, integrating these two patterns of static culture and dynamic culture into a new pattern, successfully solving some problems of microcarrier culture cells. The present invention carries out cell culture experiments with this new pattern and these three different experimental schemes of static culture and dynamic culture, and compares them, thereby verifying the feasibility and advantages of this new pattern. Under this pattern, cells adhere to the microcarriers, and the microcarriers are supported by the three-dimensional network material in the system. Similar to dynamic culture, the microcarriers are suspended in the system, without aggregation or precipitation, and are evenly distributed and remain in a static state.

[0124] In some embodiments, the cell is a mammalian cell.

[0125] 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.

[0126] In some embodiments, the stem cells are embryonic stem cells, umbilical cord blood stem cells, mesenchymal stem cells, or induced pluripotent stem cells (iPS). The mesenchymal stem cells are, for example, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, skin mesenchymal stem cells, etc.

[0127] 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.

[0128] The composition is in the form of being combined together, or in the form of being combined, in the latter case, the self-assembling peptide and other components are placed in different containers respectively.

[0129] 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.

[0130] 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, etc.

[0131] In the ninth aspect, the present invention 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, in one or more of 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 dispersants 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 cosmetics, regenerative medicine and tissue regeneration.

[0132] The three-dimensional network material of the present invention is safe and convenient during preparation, and does not require adjustment of the system's temperature, light, salt or ion components. Self-assembly can be further promoted and enhanced by being dissolved in a neutral solution system including complete cell culture medium, 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 cell adhesion in application scenarios such as cell culture and stem cell implantation, while simultaneously performing a functional simulation of the real extracellular matrix in the cell 3D culture chamber.

[0133] The responsive self-assembling peptides provided by the present invention, and the three-dimensional network systems prepared therefrom, can be used for in vitro three-dimensional culture and storage, establishing cell models, loading cells, organs, or organoids, and injecting them into animals or humans for tissue repair; they can also be used as wound dressings, hemostatic materials, etc.; or as carriers for sustained-release drugs or functional factors; or as cell preservation materials for biotherapy, tissue engineering, and regenerative medicine. In summary, the self-assembling peptides of the present invention, and the three-dimensional network systems prepared therefrom, have a wide range of applications and are safe and convenient. DETAILED DESCRIPTION

[0134] The present invention will be further described below with reference to specific embodiments. These embodiments are provided for illustration only and do not limit the scope of protection of the present invention.

[0135] The present invention provides self-assembling peptides and a hydrogel obtained by self-assembly of the self-assembling peptides under neutral pH conditions. The hydrogel-formed scaffold material has a three-dimensional network-like scaffold structure. The self-assembling peptides of the present invention can self-assemble in a neutral liquid state without the need for exogenous components, thus avoiding the risks associated with the introduction of other exogenous substances, such as certain metal salt ions and specific proteins.

[0136] 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 can still form a 3D stereoscopic distribution due to the strong cell support function, and the viscosity is low, thereby maintaining the convenience of operation when 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 self-assembly of the self-assembling peptide molecules through hydrophobic interaction and hydrogen bonding, and ultimately form a nano three-dimensional network structure, and at high concentrations (1%), a hydrogel can be formed. 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 a stable and orderly arrangement. Furthermore, aspartic acid (D) in the hydrophilic domain is a hydrophilic amino acid, which tends to be exposed in solution. The β-turn structure reduces interference from other side chains near the aspartic acid. Therefore, the β-turn structure facilitates the function of acidic amino acids. The β-turn structure makes the side chain groups of the aspartic acid on the self-assembling peptide more active, exacerbating intermolecular repulsion, affecting the molecular arrangement, and further increasing the difficulty of the self-assembling peptide forming a three-dimensional network scaffold structure. Furthermore, when the self-assembling peptide molecules approach each other during movement, they are accelerated by electrostatic interactions, increasing the kinetic energy of the entire system. Therefore, under neutral conditions, in a solution containing only the self-assembling peptide, the distribution of the self-assembling peptide molecules is relatively chaotic, and a three-dimensional network material cannot be formed. However, once the solution of such a self-assembling peptide has a pH close to its isoelectric point, most of the self-assembling peptide molecules are in a state of equal overall positive and negative charges. The intermolecular electrostatic repulsion is reduced, and hydrophobic interactions and hydrogen bonds lead to the orderly aggregation and regular distribution of the molecules, forming a three-dimensional network material.

[0137] The concentration of the self-assembling peptide in the hydrogel of the present invention is preferably greater than 0.1 wt%, preferably within a concentration range of 0.1-4 wt%, and more preferably within a concentration range of 0.1-1 wt%. The self-assembling peptide of the present invention responds rapidly to pH. When the concentration of the self-assembling peptide is greater than 1.5%, the self-assembling peptide and the hydrogel self-assemble into a hydrogel through non-covalent cross-linking, thus achieving gelation, 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.

[0138] The self-assembling peptides of the present invention not only have the advantages of rapidly assembling to form hydrogels in a short period of time, but also exhibit the properties of shear thinning and rapid recovery to their original state after removal of external forces. This indicates that under the action of strong external mechanical forces, some non-covalent bonds within the hydrogel will break, macroscopically manifesting as the gel state being destroyed and becoming a solution-like state. When the mechanical force disappears, these broken non-covalent bonds will reconnect, macroscopically manifesting as the damaged gel self-repairing and returning to a state similar to its original gel state. Furthermore, the hydrogel recovery time does not exceed 10 minutes, and the storage modulus after recovery is at least 70% of the original value, and can even reach over 85% or even over 95% of the original value. Furthermore, the hydrogels of the present invention retain the ability to self-repair after undergoing multiple shear thinning events, meaning that shear thinning only temporarily disrupts the three-dimensional network structure within the hydrogel, giving them the potential to form a gel in situ after injection. Shear thinning can be performed using a variety of mechanical forces that can exert shear or shear stress on the hydrogel, such as pipetting, centrifugation, shaking, injection, spraying, and filtration. Furthermore, the hydrogels of the present invention offer the advantage that the self-repair process after shear thinning can be terminated by dilution. Specifically, after shear thinning under mechanical force, dilution with a solvent prevents the hydrogel from returning to a gel state, rendering it liquid. This facilitates the separation of substances encapsulated within the hydrogel. For example, after culturing, storing, and transporting cells in a hydrogel, the cells can be separated from the hydrogel by shear thinning or sufficient dilution for harvesting.

[0139] 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 in use, and can be used for various purposes such as tissue repair, in vitro 3D cell culture, storage of cells and viruses, cell migration testing, organoid construction, preparation of wound dressings, preparation of scaffolds required for tissue engineering, etc.; and it supports use in the form of injection; when used in vitro, it is beneficial to the replacement of cell culture fluid and cell separation; when used in vivo, it can achieve in situ repair in the body and in vivo drug delivery, etc. For in vitro 3D cell culture, there is no need to add other components other than the complete cell culture medium; for some immune applications, it can avoid introducing other immunogenic components to interfere with the experiment. At the same time, the hydrogel of the present invention does not change the temperature and pH of the entire system during the self-assembly process and / or after the gel is formed, which brings great safety guarantees for its application in the biomedical field and can be widely used in the biomedical field.

[0140] In some embodiments, the hydrogels of the present invention are used for three-dimensional cell culture. The self-assembling peptide solution of the present invention is directly mixed with complete cell culture medium containing cells and then transferred to a culture medium such as a culture dish, culture flask, or cell culture plate to achieve three-dimensional cell culture. The environmental conditions and culture medium required for three-dimensional cell culture are consistent with those for traditional two-dimensional cell culture, eliminating the need for expensive additional equipment. The hydrogels of the present invention are suitable for the three-dimensional culture of various cell types.

[0141] In some embodiments, the hydrogels of the present invention are used to construct organoids. The self-assembling peptides of the present invention can stably support the formation of 3D cell distributions. Furthermore, under the influence of specialized organoid culture media, functional structures with preliminary organoid morphology are formed. These structures can be used to culture organoids derived from stem cell differentiation, such as primary tumor cells, induced pluripotent stem cells, and embryonic stem cells, as well as organoids derived from other primary cells besides tumors. These structures can be used for drug screening, animal model construction, and other applications.

[0142] Glossary:

[0143] Hydrogel is a hydrophilic polymer material that can form a three-dimensional network structure through chemical or physical crosslinking. Hydrogel materials can come from natural and synthetic sources. Natural polymers such as chitosan, alginate, hyaluronic acid (HA), collagen, gelatin, etc. have the advantages of being biodegradable and carrying integrin binding sites, but they are immunogenic. Synthetic polymers such as polyethylene glycol (PEG), polyacrylamide (PAM), polyvinyl alcohol (PVA) and polymethyl methacrylate (PMMA) have the advantages of strong mechanical properties, customizability, and low immunogenicity, but lack inherent biological functions and must undergo important post-processing to induce the desired response in vivo.

[0144] Hydrogels have the following properties:

[0145] 1. Good biological relevance: The polymer contains a large number of hydrophilic groups, which can absorb water dozens of times more than its own amount, and has the characteristics of swelling but not dissolving in water, and has good water retention capacity;

[0146] 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;

[0147] 3. Biodegradability: Some natural polymer materials are biodegradable, preventing secondary damage caused by implant removal. It is these unique advantages that make hydrogels shine in biomedical materials.

[0148] 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.

[0149] Peptide Self Assembly is a short chain of amino acids with a polar domain. When dissolved in a neutral solvent and physiological salt concentration, these self-assembling peptides spontaneously assemble into hierarchical nanostructures through hydrogen bonds, ionic bonds, hydrophobic interactions or van der Waals forces. Materials derived from these assemblies have the advantages of being non-toxic, non-immunogenic, non-thrombogenic, degradable and easily metabolized. At the same time, nanofibers have the same size scale as natural ECM fibers and can be easily designed to mimic the stiffness of various soft tissues. They can also be further functionalized by the attachment of cell-interacting peptide domains or cytokines and growth factors. Therefore, they can be used to design biologically relevant culture environments and improve the control of proliferating cell populations.

[0150] Self-assembling peptides can be used as drug carriers in hydrogels for wound treatment and as peptide nanofibers for cancer treatment, enabling the sustained release of small molecules, growth factors, and monoclonal antibodies. For example, self-assembling peptides are being used to promote angiogenesis in regenerating tissues and to repair skin wounds using peptide-based scaffolds. Therefore, self-assembling peptides have applications in the field of injectable therapeutic hydrogels.

[0151] The alternating hydrophilic and hydrophobic amino acid residues in the self-assembling peptides allow them to retain large amounts of water and thus form hydrogels. The side chains of the hydrophilic residues can directly interact with water, with water molecules forming inclusion complexes to surround the side chains of the hydrophobic residues. The number of hydrophobic and hydrophilic residues in the self-assembling peptides requires clever design and proportioning. If there are too many hydrophobic residues, the self-assembling peptide will be insoluble in water and precipitate out of the water; on the other hand, if there are too many hydrophilic residues, the self-assembling peptide will be highly water-soluble and therefore unable to form a hydrogel. In addition, it is also necessary to precisely and cleverly induce the peptide molecules to self-assemble into ordered nanostructured materials, such as nanofibers, nanotubes, and nanovesicles.

[0152] "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 (Ala, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W), tyrosine (Tyr, Y); valine (Val, V); selenocysteine ​​(Sec, U); hydroxyproline (Hyp, O).

[0153] In the present invention, a "peptide" is an amino acid chain. In particular, a peptide is 2 to 40 amino acids in length.

[0154] In the present invention, "self-assembly" refers to the aggregation of self-assembling peptides into an ordered structure under normal environmental conditions.

[0155] In the present invention, "β-fold" refers to a relatively extended periodic folded zigzag main chain conformation in a polypeptide chain, which is arranged in parallel or antiparallel, thereby forming a β-fold (sheet). The parallel or antiparallel conformation is determined based on the direction of the peptide arrangement from N to C terminus. Parallel arrangement means that the peptide chains are arranged from N to C terminus. Antiparallel arrangement means that the peptide chains are arranged in opposite directions (i.e., the first peptide chain is arranged from N to C terminus, and the opposite second peptide chain is arranged from C to N terminus). The parallel arrangement may include the two ends of the peptide being staggered with each other due to peptide translation. At least half of the length of the peptide is involved in the interaction force between peptides. In the antiparallel arrangement, the polypeptides are usually arranged in a line to provide two flush endpoints. This is a typical end-to-end complementary peptide.

[0156] A β-turn is an irregular secondary structure in proteins that causes a change in the direction of the polypeptide chain. β-turns often occur at the corner where the peptide chain makes a 180° turn. A β-turn consists of 3-5 amino acid residues, with the second residue being either proline (P) or hydroxyproline (O). A β-turn motif generally refers to a turn structure stabilized by hydrogen bonds between the carbonyl oxygen atom of the nth amino acid residue and the amide proton of the n+3th amino acid residue. β-turns, also known as β-elbows, reverse elbows, or β-loops, serve to connect β-strands.

[0157] The term "hydrophobicity" in the present invention refers to a property of tending to repel water or being completely insoluble in water.

[0158] 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.

[0159] Hydrophilic amino acids, also known as polar amino acids, have polar R groups that can generally form hydrogen bonds with water molecules, thus having a certain affinity for water molecules. Hydrophilic amino acids include: S, T, Y, C, U, N, Q, D, E, O, R, K, and H.

[0160] Hydrophobic amino acids, also known as non-polar amino acids, have non-polar R groups and have low or no affinity for water molecules, but have a high affinity for fat-soluble substances. They include: G, A, V, L, I, P, M, F, and W.

[0161] "Nanostructure" refers to structures with nanometer dimensions. Nanostructures can be any one-dimensional, two-dimensional, or three-dimensional shape, including nanofilms, nanofibers, nanorods, nanowires, nanofiber networks, nanospheres, nanohelices, and mixtures thereof. A nanostructure's surface has a one-dimensional structure at the nanoscale, meaning the surface thickness of the object is between 0.1 nm and 100 nm. Nanotubes have two nanometer dimensions, with diameters ranging from 0.1 to 100 nm and lengths potentially exceeding. Spherical nanoparticles have three nanometer dimensions, meaning the particle's size in each spatial dimension is between 0.1 and 100 nm.

[0162] 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.

[0163] 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.

[0164] The RGD sequence is a tripeptide sequence containing arginine (R)-glycine (G)-aspartic acid (D). It is the recognition site for integrins and their recognition proteins and is widely present in organisms. Among them, adhesion proteins in the extracellular matrix (ECM) and blood are the most common proteins containing RGD sequences 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 signal transduction function, thereby mediating many important life activities.

[0165] Microcarriers are typically non-toxic, non-rigid microspheres or microslides of uniform density used in cell culture. They enable attachment-dependent cells to grow as a monolayer by attaching to the surface of the particles during suspension culture, thereby increasing the area for cell attachment and growth, facilitating large-scale cell culture and collection. Microcarriers play a significant role in the field of large-scale in vitro cell proliferation. Based on their shape, microcarriers can be mainly divided into spherical and sheet-like shapes, with sizes ranging from approximately 100-500μm. The raw materials used to prepare microcarriers include glass, polystyrene plastic, acrylamide, dextran, gelatin, sodium alginate, polylactic acid, chitosan, etc. The surface can be modified to be charged; the density is generally controlled to be 3%-4% higher than water or lower, and suspension can be achieved with only slight agitation, and solid-liquid separation can be achieved through inexpensive methods such as centrifugation or sedimentation. Therefore, currently common commercial microcarriers can be categorized by their raw materials into cross-linked dextran microcarriers, cellulose microcarriers, protein microcarriers, and microcarriers based on synthetic polymers. Cross-linked dextran microcarriers, exemplified by the Cytodex series, are surface-modified with denatured collagen or diethylamineethyl (DEAE), resulting in an overall positive charge that facilitates cell adhesion and is therefore a widely used microcarrier. Cellulose microcarriers, exemplified by Cytopore, are positively charged through DEAE modification and possess a porous structure. This provides surface area for cell adhesion and protects cells from shear damage. Protein microcarriers are often based on gelatin or denatured collagen. For example, the Tantti 3D cell culture scaffold, made from natural collagen, has a three-dimensional, macroporous structure and is suitable for in vitro cell culture. In addition to the aforementioned natural polymers, microcarriers made from other synthetic materials, such as glass and polystyrene, offer high reproducibility. However, their applications are limited by the lack of surface sites for cell recognition and adhesion. Existing commercial microcarriers are primarily used in the biomedical and cell culture fields, with limited applications in the food industry. When selecting microcarriers for in vitro cell culture, in addition to considering conventional parameters such as density and surface properties, which influence cell attachment and proliferation, special requirements must be met, such as detachability or biodegradability. Otherwise, additional separation steps are required after culture completion, increasing process complexity and production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] Figure 1 shows the macroscopic rheological states of the self-assembling peptide of the present invention (SEQ ID NO: 2 as an example) under different pH conditions: A: pH = 2; B: pH = 6; C: pH = 6.4; D: pH = 7; E: pH = 9; F: pH = 12.

[0167] Figure 2 shows the macro-rheological state of 1.0 wt% of the self-assembling peptide of the present invention (SEQ ID NO: 2 as an example) in response to 3 mM calcium chloride (A), 3 mM magnesium chloride (B), and 1.0 wt% bovine serum albumin (C) under neutral conditions (pH = 7).

[0168] FIG3 shows a comparison of 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).

[0169] FIG4 shows TEM images of self-assembling peptides of SEQ ID NO: 1 (A), SEQ ID NO: 13 (B), and SEQ ID NO: 22 (C).

[0170] FIG5 shows 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).

[0171] FIG6 shows the rheological states of SEQ ID NO: 25 in deionized solution at pH=3, pH=6.7, and pH=12.

[0172] FIG7 shows the infrared spectra of SEQ ID NO: 25 in deionized solutions of pH=3, pH=6.7, and pH=12 (A), PBS buffer, α-MEM medium containing 10% calf serum, and pure calf serum (B).

[0173] FIG8 shows the fluorescence spectrum responses of thioflavin at concentrations of SEQ ID NO: 29 of 100 μM, 200 μM, and 400 μM, respectively.

[0174] Figure 9 shows the supporting function of SEQ ID NO: 2 and SEQ ID NO: 3 on red blood cells; 4 parallel vials were respectively 0.1wt%, 0.05wt%, 0.01wt% of 2mL of self-assembling peptide pure water solution (containing 10% calf serum and 10 8 of red blood cells) and blank group (containing no self-assembling peptide solution, 10% calf serum and 10 8 of red blood cells).

[0175] Figure 10 shows the rheological properties of 0.3wt% (A), 0.5wt% (B), and 1wt% (C) SEQ ID NO: 10 self-assembling peptide stock solutions in DMEM basal medium, where G' is the storage modulus and G" is the loss modulus.

[0176] FIG11 shows the rheological properties of 0.3 wt% (A), 0.5 wt% (B), and 1 wt% (C) self-assembling peptide stock solutions of SEQ ID NO: 10 in DMEM complete medium containing 10% calf serum.

[0177] FIG. 12 shows 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.

[0178] FIG13 shows the injectability verification of 2 wt % of the self-assembling peptide of SEQ ID NO: 20 in PBS solution.

[0179] FIG14 shows the shear stress and self-repair characteristics of 1 wt % SEQ ID NO: 24 self-assembled into a gel at pH = 6.4.

[0180] FIG15 shows the changes in storage modulus (G′) and loss modulus (G″) of 0.3 wt.%, 0.5 wt.%, and 1 wt.% self-assembling peptide solutions of SEQ ID NO: 9 at different frequencies.

[0181] FIG16 shows the zeta potential detection and pH response of the self-assembling peptide of SEQ ID NO: 29.

[0182] FIG17 shows SEM imaging of a 0.5 wt % self-assembling peptide solution of SEQ ID NO: 24 forming a stable hydrogel after freeze-drying.

[0183] FIG18 shows that a 0.5 wt % self-assembling peptide solution of SEQ ID NO: 24 formed a stable hydrogel and then liver cancer cells were cultured on the hydrogel.

[0184] FIG19 shows the supporting effects of SEQ ID NO: 9 at final concentrations of 0 (control), 0.1 wt%, 0.3 wt%, and 0.5 wt% on liver cancer cells.

[0185] FIG20 shows the effect of (2D) and (3D) the self-assembling scaffold solution of SEQ ID NO: 22 on the cell survival of mouse mesenchymal stem cells during refrigerated storage.

[0186] Figure 21 shows that 0.1wt.%, 0.3wt.%, and 0.5wt.% of the self-assembling peptide of SEQ ID NO: 9 were added to the cell culture system to induce their self-assembly into a fiber network scaffold and culture liver cancer cells. The cell sphere diameters were counted on the 3rd day, 7th day, 11th day, and 15th day, and the characteristic cell spheres in the culture system were photographed.

[0187] Figure 22 shows the imaging observation of cultured liver cancer cells after staining with Calcein AM under a laser confocal microscope. A is Calcein AM live-dead staining, and B is EDU staining.

[0188] FIG23 shows the use of the self-assembled mesh scaffold of SEQ ID NO: 21 to culture porcine muscle satellite cells under liquid conditions.

[0189] FIG24 shows the cell morphology of primary esophageal cancer organoids cultured with SEQ ID NO: 21 after 7 days.

[0190] Figure 25 shows the support effect of the self-assembling peptide solution of SEQ ID NO: 23 on polylactic acid microspheres (A) and polystyrene microspheres (B). The left image in A and B shows the microsphere solution without 0.1 wt% of the self-assembling peptide, while the right image shows the microsphere / self-assembling peptide solution with 0.1 wt% of the self-assembling peptide.

[0191] Figure 26 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 days 1, 3, 5, and 7, respectively.

[0192] Figure 27 shows that pig muscle satellite cells cultured in a polystyrene microsphere / self-assembling peptide composite system were stained with Calcein-AM / PI dye and then examined using a laser confocal microscope. A, B, C, and D represent cells on days 1, 3, 5, and 7, respectively.

[0193] FIG28 shows 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.

[0194] FIG29 shows the cell proliferation curve and cell viability characterization results of porcine muscle satellite cells cultured in the polystyrene microsphere / self-assembling peptide composite system.

[0195] FIG30 shows crystal violet-stained micrographs of HepG2 cells and Hela cells that grew adherently on a well plate after migration.

[0196] FIG31 shows the livers implanted with tumors in situ, which were dissected from the experimental group (A) and the control group (B) after 18 days of culture.

[0197] Figure 32 shows the comparison of tumor proliferation between the experimental group and the control group after N87 cells were subcutaneously implanted into SCID Beige mice. A shows the change in tumor volume, and B shows photos of the experimental group and the control group on day 18.

[0198] Example

[0199] The present invention is further explained below through specific examples. Since the present invention relates to the principle of self-assembly of RGD-containing self-assembling peptides to form three-dimensional network materials, in multiple embodiments, different self-assembling peptide sequences are used as examples to provide verification data for the hydrogel properties and functions of the present invention. It is understood that other self-assembling peptides that conform to the molecular structure of the self-assembling 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 represented by single-letter abbreviations, which are generally accepted in the technical field.

[0200] Example 1. Synthesis of self-assembling peptides

[0201] The self-assembling peptide of the present invention was synthesized by a standard solid-phase peptide synthesis method. The amino acid sequence of the self-assembling peptide is shown in SEQ ID NO: 1-31 in the sequence listing. It was identified as the target self-assembling peptide by high performance liquid chromatography and mass spectrometry. The self-assembling peptide powder was obtained after freeze-drying and stored at -20°C for drying.

[0202] Example 2. Preparation of self-assembling peptide solution

[0203] Weigh an appropriate amount of self-assembling peptide and dissolve it in deionized water. If a small amount remains undissolved, add a small amount of alkaline solution, such as sodium hydroxide, to dissolve it. After dissolution, check the pH of the self-assembling peptide solution to ensure it is between 7.0 and 7.2. Prepare a 2 wt% stock solution of the self-assembling peptide, sterilize it by autoclaving, and refrigerate it at 4°C. Prepare enough stock solution to ensure that the same batch of self-assembling peptides is used in the same experiment.

[0204] Example 3: Verification of pH, metal ion and protein responses of self-assembling peptides

[0205] Experimental method: 2wt% and 1wt% self-assembling peptide solutions were prepared under different pH environments: Taking SEQ ID NO: 2 as an example, deionized water was added to dissolve the lyophilized powder to prepare a 2wt% self-assembling peptide mother solution; sodium hydroxide and hydrochloric acid were used to adjust the pH to make the concentration of the self-assembling peptide solution 1wt% and the pH values ​​reach 2, 6, 6.4, 7, 9, and 12 respectively. After standing for 24 hours, the solution was observed and photographed.

[0206] When the self-assembling peptide solution was neutral (pH = 7), the response of the self-assembling peptide solution to metal ions (magnesium ions and calcium ions as examples) and proteins (BSA as an example) 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 of the self-assembling peptide solution to 7 and a concentration of 2 wt%. An equal amount of 6 mM magnesium chloride or calcium chloride, or an equal amount of 2 wt% BSA aqueous solution was added, respectively, so that the concentration of the self-assembling peptide in the final system was 1 wt%, the concentration of the metal ion was 3 mM, or the concentration of BSA was 1 wt%. After standing for 24 h, the system was observed and photographed.

[0207] Experimental results: When the concentration of the self-assembling peptide is 1%, it is liquid under acidic conditions (pH = 2 and 6); at pH 6.4, it is a gel peptide and does not flow when placed at an angle; at pH 7.0, it is a relatively viscous liquid, and at pH = 9.0 and pH = 12.0, it is in a liquid state (as shown in Figures AF in Figure 1). By comparison, it was found that: SEQ ID NO: 4 was liquid under acidic conditions (pH = 3); it was liquid at pH 6.0 with enhanced viscosity; it was liquid at pH 12.0 with 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, completely destroying the sequence basis for forming the β-turn region. This example proves that the β-turn region is a necessary condition for the self-assembly of the self-assembling peptide of the present invention; while SEQ ID NO: 30 (missing the RGD sequence) was a colloid at pH 2, 6, and 6.4, and liquid at other pH environments, verifying that the RGD sequence is the second necessary condition to ensure self-assembly under weak acid and neutral conditions; while SEQ ID NO: 31 (missing the RGD sequence) exhibited a turbid solution state and could not be completely dissolved, which again proved that RGD is involved in the function of the self-assembling peptide of the present invention.

[0208] In the responsiveness test of the polypeptide sequence of SEQ ID NO:2 to metal ions and proteins, it was found that when 3mM magnesium ions and 1wt% BSA were added to a 1wt% neutral (pH=7) self-assembling peptide solution, the solution did not start to form a significant hydrogel within 30 minutes; however, when calcium ions were added, the self-assembling peptide underwent rapid self-assembly and immediately became a weaker hydrogel (as shown in AC in Figure 2). By comparison with SEQ ID NO:30, it was found that the above factors can all start SEQ ID NO:30 to form a stable hydrogel. The difference between SEQ ID NO:2 and SEQ ID NO:30 is that the end of the hydrophilic end of the polypeptide in SEQ ID NO:2 is replaced by the amino acid sequence GGE with the amino acid sequence RGD. This test proves that this replacement can significantly change the self-assembly properties of the polypeptide. Comparing SEQ ID NO: 2 with 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 elicit a response. This experiment once again demonstrated the important role of RGD in the self-assembly function of the polypeptide.

[0209] 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 3A). When the concentration of the self-assembling peptide of the present invention in PBS buffer was increased to 2 wt%, the mixed solution exhibited a gel with relatively high elasticity and viscosity (Figure 3B). This verification further demonstrated that the RGD sequence completely altered the self-assembly properties of peptides without the RGD sequence. In other words, the peptides of the present invention containing RGD can undergo self-assembly without the obvious triggered assembly response of metal ions and proteins.

[0210] The responses of the self-assembling peptides shown in each sequence to different pH, metal ions and proteins are shown in Table 1.

[0211] Experimental conclusion: The results in Figure 1 and Table 1 show that 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, that is, physiological pH conditions, resulting in 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 shows 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, a comparison found that self-assembling peptides that cannot form a β-pleated structure cannot trigger hydrogel formation (such as SEQ ID NO: 5); SEQ ID NO: 30 has two β-turns, and SEQ ID NO: 31 contains only one β-turn. Both polypeptides lack the RGD sequence, but they exhibit 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 the initiating 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 peptides of the present invention to perform specific functions.

[0212] Example 4: Structural Characterization and Self-Assembly Mechanism Study of the Mesh Scaffold

[0213] Experiment 1: Transmission Electron Microscopy (TEM)

[0214] 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 %.

[0215] Preparation of self-assembling peptide samples: The 0.2wt% self-assembling peptide solution obtained in Example 2 was diluted to 0.002wt% with ultrapure water and incubated at 37°C for one hour. 10μL was placed on a 300-mesh carbon support film copper grid (purchased from Beijing Xinxing Bairui Technology Co., Ltd.) and allowed to stand at room temperature until completely dry. After complete drying, 10μL of 2wt% phosphotungstic acid negative staining solution was dropped onto the copper grid supporting the sample. After staining for 60 seconds, the excess stain was removed and the staining step was repeated three times. After staining, the copper grid was allowed to stand at room temperature to dry.

[0216] Experimental results: TEM can better see the secondary structure of the self-assembling peptide after assembly. All samples were observed and photographed using a Talos G2 200X transmission electron microscope. Taking SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 22 as examples, the imaging results are shown in Figure 4: A, B, and C in Figure 4 are TEM images of the self-assembling peptides taken with SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 22 at a concentration of 0.002 wt%. TEM can observe the network structure inside the self-assembling peptide. It can be seen from the figure that the self-assembling peptide of the present invention can self-assemble at low concentrations without an excitation substance. The nanofiber length is several hundred nanometers and the width is about 4 nm. 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 helpful in supporting cells during cell culture and creating a three-dimensional growth space for cells.

[0217] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0218] Experiment 2: Scanning Electron Microscopy (SEM)

[0219] 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) to a dilution concentration 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 be completely frozen, then frozen under liquid nitrogen for 15 minutes, and then completely freeze-dried in a vacuum freeze dryer for 24 hours. 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, the sample surface needs to be sprayed with gold to increase the conductivity of the sample. At the same time, the pore size statistics of the photographed images were performed using Image J.

[0220] Experimental results: 1wt%, 2wt%, and 3wt% samples all showed a porous structure. In the complete culture medium, the self-assembling peptides were interconnected and highly connected, forming a honeycomb-like pore structure. This structure is suitable for the living environment of cells and can achieve the transport of nutrients and oxygen. At the same time, the self-assembling peptides of the present invention contain RGD (arginine-glycine-aspartic acid) sequences, which are present 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-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. Taking Figure 5 as an example, the microstructure of the freeze-dried matrix of SEQ ID NO: 24 at a concentration of 1% (A), 2% (B), and 3% (C) is presented.

[0221] Experiment 3: Infrared (FTIR) analysis

[0222] Experimental method: Taking SEQ ID NO: 25 as an example, dissolve the self-assembling peptide in deionized water to 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 self-assembling peptide concentration 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. After testing, the pH is 7.2. The total volume of the sample is 200μL, the final concentration of the self-assembling peptide is 1wt%, and it is quick-frozen in liquid nitrogen for 15min and then freeze-dried. 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 potassium bromide pellet method were acquired 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.

[0223] Experimental results: The states of SEQ ID NO: 25 in deionized solution at pH 3, 6.7, and 12 are liquid, gel peptide, and liquid, respectively (Figure 6). From the infrared images (AB in Figure 7), it can be seen that the self-assembling peptide solution at pH = 6.7 has a higher concentration of 1670 cm-1 than the self-assembling peptide solution at pH = 3 or 12. -1 Most of the β-rotation structure peaks disappear at 1634 cm -1The peak at 1634 cm is sharper, indicating that more β-folded structures have been formed. -1 ); infrared images of the peptides stimulated with PBS, α-MEM (10% FBS), and FBS show similar changes in secondary structure. Stimulation with 10% serum alone has minimal effect on the secondary structure of the self-assembling peptides. This study demonstrates that β-turns play a crucial role in the self-assembly of the self-assembling peptides during scaffold formation, and that β-turns provide a favorable foundation for the formation of more β-sheet structures and a more regular and ordered fiber structure.

[0224] Experiment 4: Thioflavin fluorescence experiment

[0225] Experimental Method: Using SEQ ID NO:29 as an example, a 5 mM THT dye solution was diluted to 0.1 mM with ultrapure water. Self-assembling peptide solutions of 200 μM, 400 μM, and 800 μM were prepared with ultrapure water. The self-assembling peptide solutions were mixed with the diluted dye solution in a 1:1 ratio to give final concentrations of 100, 200, and 400 μM. The mixture was incubated in an oven at 37°C for 1 hour. A fluorescence spectrophotometer was turned on 30 minutes in advance and set to an excitation wavelength of 442 nm, an emission wavelength of 450-550 nm, and an operating voltage of 800 V. Five spectra were collected for each sample, with an excitation wavelength of 442 nm and excitation and emission slits of 5 nm and 2.5 nm, respectively. Each set was repeated five times, and the average value was calculated. Data analysis was performed using Origin.

[0226] Experimental Results: Thioflavin T (ThT) is a potential dye for quantitatively analyzing the β-sheet structure in self-assembling peptide nanofibers. Its fluorescence can reflect the presence of β-sheets in solution. To this end, experiments were conducted using different concentrations of the self-assembling peptide to vary the number of nanofibers. Figure 8 shows the self-assembling peptide after ThT fluorescence staining. Final concentrations of the self-assembling peptide were 100, 200, and 400 μM. At emission wavelengths between 450 and 550 nm, the peak ThT fluorescence values ​​varied with peptide concentration. β-sheets were present in the self-assembling peptide gel, and their content was positively correlated with peptide concentration, indicating that the self-assembling peptide is concentration-dependent. This is because an increase in peptide monomers facilitates intermolecular contacts, thereby enhancing the degree of assembly. ThT staining data from the gels indicate that, under neutral pH conditions, the supramolecular polymers formed by the self-assembling peptide exhibit a typical β-sheet structure. This experiment further demonstrates the importance of β-turn formation of β-sheets in the peptide structure for its self-assembly function.

[0227] 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

[0228] Experiment 1: Verification of amino acid sequence support for red blood cells

[0229] In this experiment, taking red blood cells as an example, the supporting effect of amino acid sequences on cells was verified, especially the supporting effect of hydroxyproline in the amino acid sequence on cells.

[0230] Experimental method: SEQ ID NO: 2 (IIIIIGSIIGPGGEGPRGD) and SEQ ID NO: 3 (IIIIIGSIIGOGGEGPRGD) self-assembling peptide stock solutions were prepared with PBS buffer at a concentration of 0.1 wt %, and sterilized at high temperature.

[0231] Using a 2 mL transparent glass bottle, 2 mL of pure self-assembling peptide solution (containing 10% calf serum) was prepared with 0.1 wt%, 0.05 wt%, and 0.01 wt% of each material, respectively, and a blank group (containing 10% calf serum but without self-assembling peptide solution) was added. 5 × 10 9 The final concentration of red blood cell stock solution was 1×10 8 cells / mL, pipetting evenly. Take photos every 4 hours to compare the red blood cell support effects of different materials. Because the hydrogel formed by the self-assembling peptides of the present invention is a water-swelling system, the low-concentration self-assembling peptide network scaffold, even in a solution state, still demonstrates the support capacity of the self-assembling peptide hydrogel.

[0232] Experimental conclusion: Among the self-assembling peptides with similar amino acid sequences, hydroxyproline has a significant effect on the cell support capacity of the hydrogel or the scaffold solution formed by the swelling of the hydrogel. As shown in Figure 9, under low concentration conditions (0.01wt%), the red blood cell support capacity of the two mesh scaffolds showed a significant decline 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; SEQ ID NO: 3 (IIIIIGSIIGOGGEGPRGD) self-assembling peptide solution showed no obvious sedimentation and stratification at 8 hours, 24 hours, 32 hours and 48 hours respectively; and SEQ ID NO: 2 (IIIIIGSIIGPGGEGPRGD) self-assembling peptide solution, although it can still support the three-dimensional distribution of red blood cells, showed stratification after 24 hours, and the stratification phenomenon gradually became obvious as time went by. 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.

[0233] Experiment 2, rheological experiment: Determination of changes in the mechanical properties of the mesh scaffold and the influence of the amino acid sequence on the self-assembly function.

[0234] 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 %.

[0235] 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.

[0236] In order to simultaneously measure the formation speed and strength of the mixed system gel, 200 μL of each group of samples was immediately taken and 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 Figures 10 and 11.

[0237] In this example, the storage modulus (G') of SEQ ID NOs: 1 to 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 FIG12 .

[0238] Experimental results: In each sample with different concentrations of self-assembling peptides, 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 all show that the storage modulus is less than 5 (AC in Figure 10). After the addition of calf serum, the storage modulus (G') was significantly enhanced. Even at a lower concentration (0.3%), the scaffold system still showed obvious gelation (AC in Figure 11). This experiment proves that the mesh scaffold system assembled by the self-assembling peptides of the present invention can be enhanced in response to serum, and the mechanical strength continues to increase with time. This unique property gives this 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. The continuously strengthened mesh scaffold continuously increases the support force for the cells in this process, realizing the 3D distribution of the cell system during the culture process.

[0239] Comparison of the storage moduli of different samples at 0.5 wt% in DMEM (Figure 12) revealed that amino acid sequence significantly influences the network structure formed by self-assembling peptides. Sequence ID NOs: 8, 9, 12, 28, and 29 exhibited higher storage moduli. These sequences share the common feature of alanine replacing glycine in the β-turn region. This result demonstrates that the presence of slightly hydrophobic alanine significantly enhances the mechanical strength of the self-assembling network.

[0240] In contrast, in SEQ ID NO: 5, the positions of G and P are reversed, and the structure of the β-turn region is destroyed, which causes SEQ ID NO: 5 to lose its self-assembly function, and therefore it has no storage modulus in DMEM complete medium; and in SEQ ID NO: 30, due to the absence of the RGD sequence, the solution is also turbid 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.

[0241] Experiment 3: Verification of the injectability of the mesh scaffold solution system

[0242] Experimental Method: Using SEQ ID NO: 20 as an example, a 2wt% self-assembling peptide solution (0.5mL) was prepared in PBS. The pH was adjusted to 7.0 with sodium hydroxide and allowed to stand at room temperature for 24 hours. The mixture was observed to form a hydrogel. The hydrogel was withdrawn with a 1mL syringe and quickly injected back into the vial to observe the effect of shear on the hydrogel's state during the transfer process.

[0243] Experimental results: As shown in Figure 13, 2wt% of the SEQ ID NO: 20 self-assembling peptide can form a hydrogel with high mechanical strength in PBS without any starting components, proving that the self-assembling peptide of the present invention can self-assemble in a neutral environment to form a network 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, thereby having fluidity and facilitating injection and transfer; after standing for about 10 minutes, the liquid sample in the bottle returns to a solid gel state. This shows that the gel assembled by the self-assembling peptide has good self-repairing 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 quickly recombine after a short period of standing, thereby driving the self-assembling peptide to form an ordered structure again, forming a network scaffold, and showing the re-formation of the hydrogel on a macroscopic scale.

[0244] Experiment 4: Rheological experiment: Determination of the self-repairing function of the mesh scaffold

[0245] Experimental method: As in Experiment 3, the self-assembling peptide shown in SEQ ID NO: 24 was dissolved in deionized water at 1.0 wt% of the self-assembling peptide, and the pH was adjusted to 6.4 with HCl. During the test, 200 μL of the mixed system was added to a 20 mm diameter panel of a MARS 60 rheometer for time scanning. Then, shear thinning was performed for 20 seconds under the conditions of a distance between the rotor and the panel of 500 μm, a shear strain of 100%, and a frequency of 1 Hz. After that, the shear thinning was stopped, the shear strain was immediately adjusted to 1%, and 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 subjected to multiple external forces causing deformation.

[0246] Experimental Results: As shown in Figure 14, the self-assembling hydrogel exhibits the ability to rapidly reassemble after deformation caused by external forces, and this property persists despite repeated disruptions. When subjected to 100% shear strain, some non-covalent bonds within the hydrogel, such as hydrogen bonds, break, causing its mechanical strength to decrease and transform into a liquid state. Upon removal of the external force, the partially disrupted nanofiber clusters rapidly converge and reassemble, rapidly regaining their original mechanical strength. Figure 14 demonstrates that the hydrogel of the present invention can still reassemble after undergoing multiple shear thinning events, and the self-assembly time and strength of the reassembled hydrogel increase with increasing shear thinning events, with only a small loss. This experiment demonstrates that the self-assembling peptides of the present invention exhibit shear thinning and self-repair properties, making them injectable. When used in cell culture, the hydrogel of the present invention allows for multiple pipetting transfers, facilitating cell aliquoting and fluid exchange. Furthermore, when used in biomedical applications, it allows for multiple injections.

[0247] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0248] Experiment 5: Mechanical stability of the mesh stent of the present invention

[0249] Experimental method: A shear thinning test was performed to verify the reassembly performance of the hydrogel of the present invention after deformation.

[0250] 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 10% calf serum.

[0251] Taking SEQ ID NO: 9 as an example, a frequency sweep experiment was performed on the gel sample using a German HAAKE MARS60 rheometer. The viscoelastic properties of the hydrogels at different concentrations and complete culture medium were examined using oscillatory rheology. The oscillation frequency sweep mode was selected. At a strain of 1%, the frequency was set from 0.1 Hz to 10 Hz. The temperature was maintained at 37°C. The changes in the storage modulus (G′) and loss modulus (G″) of the material were recorded as the frequency changed.

[0252] Experimental results: In each sample with different concentrations of self-assembling peptides, even when a higher frequency is used, the storage modulus is higher than the loss modulus, and the macroscopic appearance is a hydrogel state. This experiment proves the stability of the self-assembling peptides of the present invention to form a mesh scaffold. Even when subjected to higher shear forces, the hydrogel still exhibits higher mechanical stability (as shown in Figure 15). Combining Experiment 2 and Experiment 3 in this embodiment, 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 stronger shear forces, 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. After entering the tissue, it can quickly form a stable hydrogel package, and at the same time can withstand scenes such as bumps and slight vibrations, thereby stably functioning.

[0253] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0254] Experiment 6: Charge detection of the self-assembly structure of the self-assembling peptide of the present invention

[0255] Experimental Method: Using SEQ ID NO:29 as an example, a self-assembling peptide sample was dissolved in ultrapure water at room temperature to a final concentration of 0.25 wt.%. Appropriate amounts of 1 M HCl and 1 M NaOH were added to adjust the pH to 2.0, 4.0, 6.0, 7.0, 8.0, 10.0, and 12.0, respectively. Points where sudden changes in pH were observed were marked as protonation and deprotonation points. The zeta potential of the concentrated self-assembling peptide solution was measured using a zeta potential meter.

[0256] Experimental results: The main self-assembly principle of the self-assembling peptide of the present invention is mainly 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 network 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 the oppositely charged amino acid residues through electrostatic action, thereby reducing the electrostatic repulsion between peptide molecules, enhancing the interaction between peptide molecules and inducing self-assembly. The positive and negative detection of the Zeta potential is 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. As shown in Figure 16, when the overall environment is acidic, the carboxylic acid of the self-assembling peptide chain is uncharged, and NH2 combines with an H + Formation of NH3 + Positively charged. Because the self-assembled peptide chain is positively charged, it cannot form a gel under electrostatic repulsion. When NaOH solution is added gradually to an 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 network scaffold structure, that is, around the isoelectric point of 6.2, the self-assembling peptide solution forms a hydrogel on a macro scale. As the pH continues to rise, COO - Increased, 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.

[0257] Experiment 7: Characterization of the Self-Assembling Peptide Gels of the Present Invention and Cell Culture on the Gels

[0258] Experimental method: Taking SEQ ID NO: 24 as an example, the self-assembling peptide was dissolved in PBS to prepare a 2 wt % self-assembling peptide solution, which was sterilized and used as the self-assembling peptide mother solution.

[0259] Characterization of Response Layer Structure: Dilute a certain amount of self-assembling peptide stock solution to 0.5 wt% with complete DMEM cell culture medium. Add 30 μL of the 0.5 wt% self-assembling peptide solution to a tissue culture (TC)-treated 96-well plate. Allow to gel overnight at room temperature. Tilt the plate and discard the supernatant. Place the 96-well plate in a freezer pre-cooled to -50°C for 2 hours before freeze-drying. The freeze-dried sample was gold-sprayed before imaging.

[0260] 3D cell culture: dilute the self-assembling peptide stock solution to 1.0 wt% with an equal volume of complete culture medium. Take 30 μL of 0.5 wt% self-assembling peptide solution and add it to a 96-well tissue culture (TC) treated plate. Place it in a 37°C incubator overnight to form a gel. Tilt the 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.

[0261] Experimental Conclusion: After adding 0.5 wt% of the self-assembling peptides of the present invention to the culture wells and incubating overnight, a stable gel layer formed at the bottom of the wells. As shown in Figure 17, after lyophilization, the self-assembling peptide gel coating formed on the well plate possesses a regular, porous structure, providing a sufficient three-dimensional microenvironment for cells and well-established channels for gas-liquid exchange. This is likely due to the tight binding of the high concentration of self-assembling peptide chains, which forms a stable self-assembling peptide coating.

[0262] Experiments culturing HepG2 cells on the hydrogel layer (Figure 18) showed that hepatocellular carcinoma cells (HepG2 cells) could be supported on the hydrogel layer and subsequently grow and proliferate as spheroids. As the culture time increased, the spheroids became increasingly compact, and their diameters increased at a similar rate, indicating that HepG2 cells could proliferate at a uniform rate over the five-day culture period. This relatively uniform cell proliferation rate also indicates that the cells were in good condition during the culture period and were not damaged.

[0263] This experiment uses TC-treated culture vessels as an example to demonstrate that the self-assembling peptides of the present invention can react on the surface of culture vessels to form stable hydrogels, which can be used for 3D cell culture on the hydrogels. At the same time, this method is very easy to perform operations such as cell fluid replacement, and the hydrogels are transparent and colorless, which is also convenient for imaging characterization.

[0264] Example 6: Application of the peptide hydrogel material of the present invention in the field of 3D cell culture

[0265] Experiment 1: The supporting effect of the hydrogel of the present invention on cells

[0266] 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 high-glucose DMEM medium with 10% serum content. 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. Different volumes of 2wt% self-assembling peptide mother solution were then 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 an LSM 980with Airyscan2 fast super-resolution laser confocal microscope.

[0267] Experimental conclusion: The results in Figure 19 show that in the control group without the hydrogel of the present invention, the cells naturally sank to the bottom of the glass-bottom culture dish, were not supported, and were distributed in a 2D plane; when 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-assembling peptides, showing a three-dimensional distribution in a 3D environment.

[0268] This experimental phenomenon demonstrates that the hydrogel material of the present invention, namely, a three-dimensional network scaffold formed by the self-assembling peptides, can effectively support cells, facilitating their growth and cultivation in three-dimensional space in vitro, preventing them from sinking and adhering to a wall and subsequently growing in a two-dimensional environment. Furthermore, the entire initiation process is mild, requires no additional substances, and is effective, convenient, and harmless to cells, demonstrating the enormous potential of the self-assembling peptide material of the present invention in the fields of cell culture and biomedicine.

[0269] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0270] Experiment 2: Application of 3D Cell Storage

[0271] Experimental method: Taking SEQ ID NO: 22 as an example, this experiment uses the storage of mouse mesenchymal stem cells at 4°C as an example. The isolated mouse mesenchymal stem cells were pipetted evenly, and 10 mL of equal 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% bispecific antibody in 50% MAP + 50% SFEM storage solution, and the self-assembling peptide SEQ ID NO: 22 was added to a final concentration of 0.05 wt %. The 2D group was prepared by adding mouse serum and 1% bispecific antibody in 50% MAP + 50% SFEM storage solution, and then stored in a 4°C refrigerator.

[0272] Cell viability test:

[0273] (1) Thoroughly mix the stored cells by pipetting, take 500 μL of cell suspension into each 1.5 mL EP tube, add PBS to wash, and centrifuge at 1500 rpm for 10 min;

[0274] (2) Discard the supernatant and resuspend the cells in 150 μL of 1% PBA solution. Then add 150 μL of live-dead cell staining solution, mix thoroughly, incubate at 37°C for 15 min in the dark, and detect using flow cytometry.

[0275] 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 for detection. As shown in Figure 20, 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 relatively high (81% vs. 43%), and the survival rate was increased by nearly 50%. This shows that the storage effect of the mesenchymal stem cell storage system used in the present invention is better than traditional 2D storage, which greatly improves the cell survival rate.

[0276] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0277] Experiment 3: Application of 3D Culture of Liver Cancer Cells

[0278] 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. There are concentration gradients of oxygen, nutrients, and metabolic waste in cell spheres, which can simulate the various characteristics of solid tissues. It is an important 3D physiological model for studying the occurrence of solid tumors and stem cell differentiation, and is widely used in the field of biomedicine. Moreover, cell spheres are simpler than other 3D physiological models. During the research process, imaging analysis can be achieved through common experimental means, such as optical, fluorescence, and confocal microscopy, which simplifies the experimental process in the research. This experiment used the adherent cells HepG2 cultured in the self-assembling peptide culture system of the present invention induced by high-glucose culture medium, and whether cell spheres could be formed was used to verify that cell culture similar to that in organisms can be achieved in vitro.

[0279] Prepare the solution with a concentration of 1×10 6 The concentration of 1×10 cells / mL cell suspension was prepared by 5 cells / mL of cell suspension. Taking SEQ ID NO:9 as an example, the concentration obtained is 1×10 5 A cell suspension of 10 cells / mL was inoculated into a 96-well plate 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 0.1 wt%, 0.3 wt%, and 0.5 wt%). During the culture period, the culture medium in the upper part of the plate was removed and replaced with new culture medium every 3 days.

[0280] The diameters of 50 cell spheres in the culture system were observed and recorded under an inverted microscope on the 3rd, 7th, 11th, and 15th day of culture. 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 characteristic cell spheres were photographed.

[0281] In the EDU fluorescence 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 the digestion, and the culture flask was rinsed with complete medium to remove the remaining cells. The cells were centrifuged at 800 rpm for 3 min, and counted using a cell counting plate. The cell suspension was diluted to 5×10 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 kit on the 3rd, 7th, 11th, and 15th day of culture, respectively, and photographed under a confocal fluorescence microscope.

[0282] Test results: As shown in Figure 21, in a culture system containing 0.1wt.%, 0.3wt.%, and 0.5wt.% of the triggered self-assembling peptide of the present invention, HepG2 cells were supported and subsequently grew and proliferated in the form of cell spheres. As the culture time prolonged, the cell spheres became increasingly compact, and their diameters increased at a similar rate. This indicates that HepG2 cells can stably proliferate at a uniform rate over a 15-day culture period. This indicates that the culture system containing the triggered self-assembly peptide of the present invention can be used for long-term in vitro three-dimensional cell culture. The relatively uniform cell proliferation rate also indicates that the cells are in good condition during the culture period and are not damaged.

[0283] Brightfield images demonstrate that the self-assembling peptides promote cell aggregation. Therefore, confocal fluorescence microscopy was used to observe the state of the cell spheroid culture and whether it was proliferating. As shown in Figure 22A, the activity of the cell spheroids is increasing. Most of the HepG2 spheroids cultured in the presence of the self-assembling peptides of the present invention are calcein AM positive, with very few dead cells. This confirms the high cell viability and absence of necrotic cores during the culture process. This also demonstrates that the self-assembling peptides of the present invention can provide a very suitable growth environment for cell growth and survival by mimicking the extracellular matrix, thereby achieving optimal in vivo culture results.

[0284] The proliferation of HepG2 spheroids cultured in hydrogels was further assessed by imaging with 5-ethynyl-2′-deoxyuridine (EDU). As shown in Figure 22B, proliferating cells were observed to be randomly distributed throughout the HepG2 spheroids, indicating that sufficient nutrient diffusion occurred through the self-assembling peptides and spheroids of the present invention during the culture process. As the number of days increased, proliferation decreased by day 15, indicating that the cells began to approach differentiation as proliferation decreased.

[0285] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0286] Experiment 4: Application of 3D Culture of Porcine Muscle Satellite Cells

[0287] Experimental method: The culture of porcine muscle satellite cells was similar to the 3D culture of liver cancer cells in Experiment 3. The 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 5 A cell suspension of 10 cells / mL was inoculated into a 96-well plate 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.%). Every 3 days during the culture period, the culture medium of the well plate was removed and centrifuged to precipitate the cells, which were then resuspended in fresh culture medium containing 0.3 wt.% and continued to be cultured.

[0288] Experimental results: After the 6th 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 can achieve long-term in vitro proliferation, and characteristic cell spheres were photographed (Figure 23). As shown in Figure 23, in the culture system containing 0.3wt% of the self-assembling peptide of the present invention, the porcine muscle satellite cells were supported and subsequently grew and proliferated in the form of cell spheres. As the culture time prolonged, the cell spheres became more and more compact, forming a high-density cell cluster with a three-dimensional distribution, indicating that the porcine muscle satellite cells proliferated well over a period of 6 days, that is, the self-assembling peptide system of the present invention formed 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 the vast majority of cells were living cells, which were in good condition during the culture period and did not cause damage to the cells.

[0289] Experiments 1-4 of this example demonstrate that the culture system of the self-assembling peptides of the present invention has excellent cell-supporting capacity. Experiment 2 also demonstrates the good biocompatibility of the self-assembling peptides of the present invention and their application in cell storage. Experiments 3 and 4 demonstrate that the addition of the self-assembling peptides of the present invention to cell culture medium can highly replicate the in vivo cell growth environment, enabling long-term three-dimensional cell culture in vitro.

[0290] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0291] Experiment 5: Construction of Organoids

[0292] 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 using the cell sieve, resuspend with F12 culture medium, centrifuge the filtrate, 250g, 5min, and discard the supernatant. Add 5mL F12 culture 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 a final cell concentration of 4×10 6 / mL. Take 200μL of the cell-hydrogel mixture and gently pipette it evenly. Then, add three drops per well to a low-adhesion 24-well cell culture plate, for a total of three wells. Then, place the cell culture plate upside down in an incubator overnight. The next day, remove the cell culture plate, add 500μL of specialized culture medium to each well, and culture in an incubator. Observe daily and change the medium every two days.

[0293] Experimental results: After 7 days of continuous culture, the primary esophageal cells can proliferate into a spherical structure with an organoid morphology (Figure 24). This experiment proves that for the patient's primary tumor cells, 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, as well as the culture application of organoids of other primary cells besides tumors.

[0294] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0295] Example 7: Application of the self-assembling peptide of the present invention as a microsphere dispersant

[0296] Experiment 1: Verification of the support of self-assembled network scaffolds for polylactic acid microspheres and polystyrene microspheres

[0297] Experimental method: The self-assembling peptide of the present invention self-assembles to generate a network scaffold that has a good dispersion support function 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, to a final concentration of 0.1wt% of the self-assembling peptide and a final concentration of 1wt% of the microspheres; 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.

[0298] Experimental Results: As shown in Figures AB and 25, the left image shows a microsphere solution without the addition of self-assembling peptides. The polylactic acid microspheres (A) and polystyrene microspheres (B) completely sank to the bottom of the bottle after 30 minutes. However, after adding 1 wt% of the self-assembling peptide (right image), the microspheres were evenly distributed in 3D space. The resulting self-assembling peptide-containing mixed solution was left at room temperature for three months, revealing a uniform suspension with no apparent sedimentation or separation.

[0299] This experimental observation demonstrates that the self-assembling peptides of the present invention can form a three-dimensional network scaffold that effectively supports microspheres in addition to cells. The microspheres provide excellent dispersion and support in the solution. The entire experimental process requires no additional components and is easy to perform. The large-diameter microspheres used in this experiment fully demonstrate the support capacity of the self-assembling peptides of the present invention to form a network scaffold.

[0300] Experiment 2: Application of Self-Assembled Network Scaffold / Microcarrier Composite System for Cell Proliferation

[0301] 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 complex system on cell proliferation. In the cell culture experiment, sterile microspheres were first added to a 48-well plate at a final concentration of 1 wt%, and then cultured porcine muscle satellite cells were inoculated at 5×10 4 Cells were added to each well of the culture plate. After incubating the cells with the microspheres for 1 day, a 2 wt % self-assembling peptide stock solution was added to a final concentration of 0.1 wt % for culture. Cells were counted on the 1st, 3rd, 5th, and 7th days. The culture medium in the wells was first aspirated, and the cells were washed 2-3 times with PBS. Then, trypsin containing EDTA was added for digestion for about 1 hour to allow the cells to fall off the carrier, and then counted using a cell counting plate.

[0302] Cell viability and cytotoxicity were assessed using the CCK8 assay. Cells were cultured on days 1, 3, 5, and 7, as described above. Cell morphology was observed using confocal microscopy, using Calcein-AM / PI staining.

[0303] 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. 2D group, microcarrier group, and 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 and no bioreactor was used. The concentration of 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 shown in Figures 26 AD and 27 AD, respectively, showing the changes in cell density on the microspheres during the proliferation of the polylactic acid microsphere / self-assembling peptide composite system and the polystyrene microsphere / 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 seventh day, the cells had proliferated significantly and wrapped around the entire surface of the microspheres.

[0304] The CCK8 assay for the proliferation and cell viability of porcine muscle satellite cells revealed that the microsphere / self-assembling peptide composite system exhibited superior results in both the comparison of cell growth density and cell viability curves (Figure 28 (polylactic acid / self-assembling peptide) and Figure 29 (polystyrene microspheres / self-assembling peptide). This experiment further demonstrates that the addition of self-assembling peptides to support the microcarriers increases the contact area between the microspheres and cells, accelerating the flow of nutrients and air, which in turn promotes cell proliferation and changes in cell status.

[0305] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0306] Example 8: Research and application of cell migration (transwell)

[0307] 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 with serum-free medium to a concentration of 1 mg / mL; 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 level and the thickness of the glue is uniform) and place the chamber in a 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 any 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 to 7×10 5 Prepare a 24-well plate containing 20% ​​serum-containing culture medium and add 600 μL per well. Use sterile forceps to gently place the chamber into the plate (avoiding bubbles). Mix the cell suspension thoroughly, then gently add 200 μL of the suspension to the chamber. Cover the plate and incubate in a 37°C incubator for 48 hours.

[0308] Experimental results: As shown in Figure 30 (photographed under a 4X microscope), the polypeptide hydrogel was 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 degraded the adjacent matrix and migrated through the ECM gel to complete the invasion, thereby adhering to the bottom of the culture well plate and proliferating.

[0309] Example 9: Application of tumor transplantation in animals

[0310] Experiment 1: Orthotopic liver tumor grafting in mice

[0311] Experimental method: This experiment uses SEQ ID NO: 22, and takes the liver in situ tumor of mice as an example to verify the application of the present 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 cut 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 hepatocellular carcinoma cells) in PBS (control group) or 30 μL containing 2×10 6 A self-assembling peptide suspension (final peptide concentration of 0.75 wt%) containing EGFP-Luc-Hepa1-6 cells was placed in the experimental group. The microinjector was slowly withdrawn, and the needle hole was sealed with medical glue. The abdomen was sutured layer by layer and disinfected again with ethanol. Mice were intraperitoneally injected with 15 mg / mL D-luciferin, anesthetized with isoflurane and oxygen inhalation, and placed in the supine position in the darkroom of the Lumina II imaging system for in situ imaging of HCC.

[0312] Experimental Results: After 18 days of culture, tumor-seeded livers from mice in the experimental and control groups were dissected and shown in Figure 31. The control group (B) showed irregular liver tumor morphology, with nodular, infiltrative growth, an uneven surface, and a hard texture. The experimental group (A) showed that after encapsulation with the peptide hydrogel, the inoculated cells formed a cohesive tumor mass, facilitating statistical analysis. In the group without the peptide, tumor cell clumps were scattered throughout the liver, hindering statistical observation and comparison.

[0313] Experiment 2: Subcutaneous tumor implantation in mice

[0314] 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 in a supine position on a mouse board, and routinely disinfected with 75% ethanol. The abdominal skin was cut horizontally, and a microinjector was inserted into the subcutaneous part of the abdomen. 30 μL containing 3×10 6 N87 cells (human gastric cancer cells) PBS suspension (control group) or 30 μL containing 3 × 10 6 A self-assembling peptide suspension (final peptide concentration of 0.75 wt%) was placed in the N87 cells (experimental group). The microinjector was slowly withdrawn and the needle hole sealed with medical glue. The abdomen was sutured layer by layer and disinfected again with ethanol. Both groups of mice were cultured for 20 consecutive days.

[0315] Experimental Results: A comparison (Figure 32, AB) revealed that subcutaneous tumor implantation in the experimental group was significantly faster than in the control group. On day 18, the diameter of the tumors in the experimental group was 30% larger than in the control group. Furthermore, the tumors in the experimental group were regular in shape, while those in the control group were irregular in shape and smaller in size. This experiment further demonstrates the utility of the hydrogels formed by the self-assembling polypeptides of the present invention as an embedding material for cells or functional components in animal models.

[0316] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention are intended to be included in the scope of protection of the present invention.

Claims

1. A 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%, performing the initiation 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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