Microsphere filler and application thereof

Abstract

The invention relates to a microsphere filler and application thereof, the microsphere filler comprises a composition containing a self-assembling peptide and microspheres, the self-assembling peptide comprises a hydrophobic structural domain and a hydrophilic structural domain, and the hydrophilic structural domain comprises at least two continuous beta-corner regions. The microsphere filler can respond and self-assemble to form a nano network structure under the condition of substances with wide sources, and can play a self-assembled peptide microsphere system with supporting and repairing functions in a solution state.

Description

Microsphere fillers and their applications Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a microsphere filler. Background Art

[0002] Microspheres are widely used in medical aesthetics as regenerative fillers. Functional microspheres, such as poly(L-lactic acid), can break the ester bonds in their molecular chains in the presence of water, producing lactic acid, which stimulates collagen regeneration in tissues. All approved medical aesthetic microsphere injectables are freeze-dried powder injections to prevent microsphere degradation during shelf life. Therefore, medical aesthetic microsphere fillers face the problem of reconstitution during clinical use. Insufficient reconstitution, microsphere adhesion, and uneven injection can all lead to adverse reactions such as subcutaneous nodules or redness and swelling. Even with extended reconstitution time or post-injection facial massage of the injection site, a certain percentage of clinical cases still experience adverse reactions such as subcutaneous nodules or redness and swelling.

[0003] Taking polylactic acid fillers as an example, polylactic acid fillers are often freeze-dried by mixing polylactic acid with sodium hyaluronate (HA) or sodium carboxymethylcellulose (CMC) to obtain a finished product. Therefore, when used, they need to be reconstituted with injection water (or normal saline) and mixed evenly before use. This results in a long reconstitution time (24 hours) during use, and it is difficult to completely reconstitute and evenly disperse the product. This can clog the needle during injection, making it more difficult for physicians to operate and detrimental to the consumer experience. This requires optimization of the preparation process of polylactic acid fillers (for example, CN 113244449 uses a small molecule fast dissolving agent-suspending agent system to prepare a polylactic acid filler that can be quickly reconstituted) to improve the product's ease of use. In addition, ordinary hyaluronic acid fillers are metabolized into CO2 and water in the body within 1-2 days. Using a cross-linking method (i.e., cross-linked hyaluronic acid), the effect is maintained for approximately 6-9 months, and the therapeutic effect of some products can last for 1-2 years. However, methods for chemically modifying microspheres and various fillers to solve the re-dissolution problem (such as the chemical reaction of polylactic acid microspheres with hyaluronic acid via carbodiimide and adipic acid dihydrazide in CN102911380 to obtain a solid polylactic acid modified particle composite gel) not only increase production costs, but also have the problem of being unable to completely remove crosslinkers and modifiers.

[0004] Self-assembling peptides are peptide molecules that self-assemble through non-covalent bonding forces such as hydrogen bonds, hydrophobic interactions, and π-π stacking to form morphologically and structurally specific peptide molecular aggregates. Self-assembling peptide molecular aggregates can form nanoscale three-dimensional network structures that can provide support for medical aesthetic microspheres (in CN114404651A, dipeptide hydrogels were used to support and load calcium carbonate particles). The network structure formed by self-assembly of self-assembling peptides is similar to the structure of the natural extracellular matrix and has ideal biocompatibility (for example, the peptide hydrogel described in CN113493492A can promote the healing of damaged skin in diabetic mice, the formation of skin appendages, increase the PCNA cell positivity rate, and promote the accumulation of CD31-positive blood vessels and collagen; CN109771694A demonstrates that self-assembling peptide nanofiber hydrogel scaffold materials can provide a reliable microenvironment for human umbilical cord Wharton's jelly mesenchymal stem cells).

[0005] Self-assembly of self-assembling peptides can be categorized as spontaneous and triggered. Spontaneous self-assembly refers to the spontaneous formation of assemblies after dissolving the self-assembling peptide in an aqueous solution. Triggered self-assembly of self-assembling peptides refers to self-assembly triggered by changes in the external environment, such as temperature (CN107365378A), pH (the palmitoyl peptide described in CN112587477A can self-assemble under acidic conditions), ion concentration (the self-assembling peptide hydrogel described in CN113493492A requires a specific ion concentration), and biomolecules (the addition of exogenous proteins to gel the peptide solution as described in CN105025942A; the self-assembling peptide hydrogel described in CN113956328A is formed by the self-assembling peptide derivatives catalyzed by alkaline phosphatase). Appropriately designed triggered self-assembling peptides can form assemblies even without triggering, providing support and dispersion for the microspheres.

[0006] Therefore, the development of microsphere fillers that can respond and self-assemble to form nano-network structures under physiological conditions and under conditions of substances widely present in the human body, and can perform supporting and repair functions in solution state will have very important application prospects.

[0007] Summary of the Invention

[0008] The present invention relates to a self-assembling peptide microsphere filler, which can respond to a wide range of endogenous material conditions and self-assemble to form a nano-network structure, and can also play a supporting and repairing role in a solution state.

[0009] In a first aspect, the present invention provides a composition comprising a self-assembling peptide and microspheres, wherein the self-assembling peptide comprises a hydrophobic domain and a hydrophilic domain, wherein the hydrophilic domain comprises at least two consecutive β-turn regions capable of forming β-turns.

[0010] In some embodiments, the at least one β-turn region comprises or is linked to one or more acidic amino acids at a terminal end, preferably comprises or is linked to one acidic amino acid.

[0011] In some embodiments, at least one β-turn region comprises an acidic amino acid at a terminal end.

[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 β-turn motif comprises one hydroxyproline (O), and preferably, X2 is hydroxyproline (O).

[0016] In some embodiments, the hydrophilic domain comprises 2-8 β-turn regions.

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

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

[0019] Preferably, the hydrophilic domain comprises 2 or 3 β-turn regions.

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

[0021] In some embodiments, the β-turn motif in the at least one β-turn region comprises or is linked to a glutamic acid (E), valine (V), leucine (L), isoleucine (I), aspartic acid (D) or lysine (K).

[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 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.

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

[0028] In some embodiments, the hydrophilic domain comprises at least one β-turn motif in which X1 is G.

[0029] 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.

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

[0031] In some embodiments, the hydrophilic domain comprises two β-turn motifs in which X2 is P.

[0032] In some embodiments, one or more of X1, X3 and X4 is glycine (G), and / or one or both of X3 and X4 is alanine (A).

[0033] In some embodiments, the β-turn motif comprises an amino acid sequence selected from the group consisting of:

[0034] GPGG (SEQ ID NO.:33), GPGA (SEQ ID NO.:34), GPAG (SEQ ID NO.:35), GPG, GPAA (SEQ ID NO.:36), GPGGG (SEQ ID NO.:37), GOGG (SEQ ID NO.:38), GOGA (SEQ ID NO.:39), GOAG (SEQ ID NO.:40), GOGGA (SEQ ID NO.:37) NO.:41), GOAA (SEQ ID NO.:42), GOG, or GOGV (SEQ ID NO.:43).

[0035] Preferably, the β-turn motif has an amino acid sequence selected from the group consisting of:

[0036] GPAGE (SEQ ID NO.:44), GPGGE (SEQ ID NO.:45), GOGAE (SEQ ID NO.:46), GOGGAE (SEQ ID NO.:47), GOGE (SEQ ID NO.:48), GOGGE (SEQ ID NO.:49), GPGAD (SEQ ID NO.:50), GOGGD (SEQ ID NO.:51), GPGGV (SEQ ID NO.:52), GOGGV (SEQ ID NO.:53), GPGGK (SEQ ID NO.:54), GOGGK (SEQ ID NO.:55), GPGAE (SEQ ID NO.:56), GOGAD (SEQ ID NO.:57), GPAAD (SEQ ID NO.:58), GOAAE (SEQ ID NO.:59), GPGGD (SEQ ID NO.:60), GPGGGV (SEQ ID NO.:61), GPGV (SEQ ID NO.:62), GOGGI (SEQ ID NO.:63) or GOGVI (SEQ ID NO.:64).

[0037] In some embodiments, X1 and X4 form a hydrogen bond.

[0038] In some embodiments, the hydrophilic domain comprises 2, 3, 4, 5, 6, 7 or 8 β-turn motifs, preferably, the hydrophilic domain comprises 2 or 3 β-turn motifs.

[0039] In some embodiments, the beta-turn motif has an amino acid sequence selected from the group consisting of:

[0040] GOGG (SEQ ID NO.:38), GPGG (SEQ ID NO.:33), GOGA (SEQ ID NO.:39), GOAG (SEQ ID NO.:40), GPGA (SEQ ID NO.:34) or GPAG (SEQ ID NO.:35).

[0041] At least one beta-turn motif in the hydrophilic domain includes an alanine, thereby improving the mechanical properties of the self-assembling peptide.

[0042] In some embodiments, the C-terminus of the hydrophilic domain can be modified with an agent or group selected from the group consisting of carboxylic acid, thiol, ketoate, nitrite, phosphonate, thiophosphate, carbonate, sulfate, nitrate, vinyl sulfone, amide, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and / or silane modifications.

[0043] The amino acid sequence of the hydrophilic domain is more hydrophilic than the amino acid sequence of the hydrophobic domain.

[0044] 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.

[0045] Preferably, the hydrophobic amino acid is selected from one or more of isoleucine (I), valine (V), leucine (L), phenylalanine (F) and alanine (A).

[0046] In some embodiments, the hydrophobic amino acid is selected from one or more of I, V, L, and F.

[0047] In some embodiments, the N-terminus of the hydrophobic domain is modified with an agent or group selected from the group consisting of acetyl, alcohol, aldehyde, amine, imine, maleimide, thiol, vinyl sulfone, azide, alkyne, olefin, ester, thioester, aryl and / or silane modifications.

[0048] In some embodiments, the hydrophobic domain has an amino acid sequence selected from the group consisting of:

[0049] LLLL (SEQ ID NO.:65), FIIII (SEQ ID NO.:66), IIII (SEQ ID NO.:67), IIII (SEQ ID NO.:68), ILILI (SEQ ID NO.:69), FLFLF (SEQ ID NO.:70), IVIVI (SEQ ID NO.:71), VLFIIV (SEQ ID NO.:72), VLIII (SEQ ID NO.:72) NO.:73), IVALF (SEQ ID NO.:74), LFIVL (SEQ ID NO.:75), FIAIV (SEQ ID NO.:76), FIIIV (SEQ ID NO.:77), Ac-VLFIIV (SEQ ID NO.:78), Ac-IVIVI (SEQ ID NO.:79), Ac-IIIIII (SEQ ID NO.:80), IIIIII (SEQ ID NO.:78) NO.:81), FLIVI (SEQ ID NO.:82), FLIIA (SEQ ID NO.:83), FIFIF (SEQ ID NO.:84), IFIFI (SEQ ID NO.:85), IAILI (SEQ ID NO.:86) or LLLLL (SEQ ID NO.:87).

[0050] The amino acid sequence of the hydrophobic domain is hydrophobic compared to the amino acid sequence of the hydrophilic domain.

[0051] In some embodiments, the self-assembling peptide further comprises a linker domain providing a spacer between the hydrophobic domain and the hydrophilic domain.

[0052] In some embodiments, the linker domain comprises 2-8 amino acid residues, preferably 4-5 amino acid residues.

[0053] 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.

[0054] In some embodiments, the amino acid with a smaller side chain is selected from glycine (G), alanine (A), and serine (S).

[0055] In some embodiments, the amino acid with a hydroxyl group on the side chain is selected from serine (S), threonine (T) and hydroxyproline (O),

[0056] 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.

[0057] In some embodiments, the connecting domain has an amino acid sequence selected from the group consisting of:

[0058] GSII (SEQ ID NO.: 88), GPOGI (SEQ ID NO.: 89), GPOGV (SEQ ID NO.: 90), GSGII (SEQ ID NO.: 91), GSVI (SEQ ID NO.: 92), GOII (SEQ ID NO.: 93), GPOGL (SEQ ID NO.: 94), OGII (SEQ ID NO.: 95) or GTVI (SEQ ID NO.: 96), wherein S, T, and O are interchangeable;

[0059] More preferably, the connecting domain has an amino acid sequence selected from the group consisting of:

[0060] GSII (SEQ ID NO.:88), GTII (SEQ ID NO.:97), GTVI (SEQ ID NO.:96), GOVI (SEQ ID NO.:98), GSVI (SEQ ID NO.:92), GSVL (SEQ ID NO.:99), GSGII (SEQ ID NO.:91), GSGVI (SEQ ID NO.:100), GOII (SEQ ID NO.:100) NO.:93), OGII (SEQ ID NO.:95), GOGVI (SEQ ID NO.:101) or GOGII (SEQ ID NO.:102).

[0061] 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.

[0062] In some embodiments, the self-assembling peptide has a length of 15-50 amino acids, preferably 15-25 amino acids.

[0063] 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.

[0064] In some embodiments, the self-assembling peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7 and SEQ ID NOs: 9-32:

[0065] IIIIIIGSIIGPGGDGPGGV (SEQ ID NO. 1);

[0066] IIIIIGSIIGPGGEGPGGV(SEQ ID NO.2)

[0067] IIIIIGSIIGOGGEGPGGV(SEQ ID NO.3)

[0068] IIIIGSIIGOGGEGPGGV(SEQ ID NO.4);

[0069] IIIIIGSIIGOGGPGGGGV(SEQ ID NO.5)

[0070] IIIIIGSIIGOGGEGPGV(SEQ ID NO.6)

[0071] IIIIIGIIGOGAEGPGGV(SEQ ID NO.7)

[0072] IIIIIGSIIGOGGVGPGGV(SEQ ID NO.9)

[0073] IIIIIIGSIGOGAEGPGGVGPGGV(SEQ ID NO.10);

[0074] FLIGHTSIIGOGAEGPGGV(SEQ ID NO.11)

[0075] FLIIAGSIIGPGGDGOGGV(SEQ ID NO.12)

[0076] IIIIIGOGIIGPGGEGPGGE(SEQ ID NO.13)

[0077] FIFIFGTVIGPGGEGOGGV(SEQ ID NO.14)

[0078] IFIFIGTVIGPGGEGOGGK(SEQ ID NO.15)

[0079] IAILIGTVIGPGGEGOGGE(SEQ ID NO.16)

[0080] IVIVIGSIIGPGGDGPGGV(SEQ ID NO.17)

[0081] IVIVIGSIIGOGGDGPGGV(SEQ ID NO.18)

[0082] IVIVIGSIIGPGGEGOGGV(SEQ ID NO.19)

[0083] FLIVIGOGIIGOGGEGPGGE (SEQ ID NO. 20);

[0084] IVIVIGIGIIGOGGDGOGGV (SEQ ID NO. 21);

[0085] IVIVIGSGIIGPGGEGPGGV (SEQ ID NO. 22);

[0086] FIIIVGSIIGPGGEGPGGV (SEQ ID NO. 23);

[0087] FIIIVGSIIGPGGEGPGGE (SEQ ID NO. 24);

[0088] IIIIIIGOGIIGOGGEGPGGV (SEQ ID NO. 25);

[0089] Ac-IIIIGSIIGPGGEGOGGV (SEQ ID NO. 26);

[0090] FLIVIGSIIGOGAEGPGGV (SEQ ID NO. 27);

[0091] FLIVIGSIIGOGAEGOGGV (SEQ ID NO. 28);

[0092] LLLLLSVLGPAGEGPAGE(SEQ ID NO.:29);

[0093] LLLLLGPOGLGPAGEGPAGE(SEQ ID NO.:30);

[0094] LLLLLGPOGVGPAGEGPAGE (SEQ ID NO.: 31); or

[0095] LLLLLGPOGIGPAGEGPAGE (SEQ ID NO.:32).

[0096] The self-assembling peptide of the present invention having the above structure can be triggered by an initiator to form a three-dimensional network scaffold material.

[0097] The three-dimensional mesh scaffold material has a nanostructure.

[0098] In some embodiments, the microspheres are selected from polylactic acid microspheres (eg, poly-L-lactic acid (PLLA) microspheres), polycaprolactone microspheres, polylactic-co-glycolic acid microspheres, hydroxyapatite microspheres, polymethyl methacrylate microspheres, and polyvinyl alcohol (PVA) microspheres.

[0099] The particle size of the microspheres is 3-150 μm.

[0100] Preferably, the particle size of the microspheres is 10-60 μm.

[0101] Preferably, the particle size of the polylactic acid microspheres is 10-60 μm, the particle size of the polycaprolactone microspheres is 10-60 μm, the particle size of the polylactic acid-glycolic acid microspheres is 20-60 μm, the particle size of the hydroxyapatite microspheres is 25-45 μm, and the particle size of the polymethyl methacrylate microspheres is 20-60 μm.

[0102] In some embodiments, the composition further comprises a positive charge source as an initiator.

[0103] The positive charge source material includes a material with a positively charged group or a positively charged ion.

[0104] In some embodiments, the positive charge source substance is a biomacromolecule, drug, functional molecule, or small molecule such as metal ions, amino acids, etc., whose number of hydrogen bond acceptors is less than the number of hydrogen bond donors under neutral conditions, or a mixture containing one or more of the above substances.

[0105] In some embodiments, the biomacromolecules include but are not limited to organic acids, proteins, polysaccharides and derivatives thereof, and the organic acids include but are not limited to lactic acid, tannic acid, citric acid and the like.

[0106] In some embodiments, the protein is selected from proteins that are capable of donating hydrogen ions under neutral physiological conditions.

[0107] Preferably, the proteins are independently selected from proteins having an isoelectric point (PI) value lower than 7.0 (preferably 3.4-6.05).

[0108] In some embodiments, the protein includes, but is not limited to, fibrinogen, globulin, hemoglobin, transferrin, laminin, fibronectin, vitronectin, and the like.

[0109] In some embodiments, the polysaccharide and its derivatives include but are not limited to chitin, chitosan, and the like.

[0110] In some embodiments, the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.

[0111] In some embodiments, the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, etc.;

[0112] In some embodiments, the antioxidants include but are not limited to vitamins such as niacinamide.

[0113] In some embodiments, the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.

[0114] In some embodiments, the metal ions include, but are not limited to, sodium, potassium, calcium, magnesium ions, and the like.

[0115] In some embodiments, the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.

[0116] In some embodiments, the positive charge source is urea or nicotinamide mononucleotide.

[0117] In some embodiments, the positive charge source substance is serum, plasma, animal or plant tissue fluid, or a mixture containing the above positive charge source substances.

[0118] The self-assembling peptide and the microspheres are placed in the same container in the form of powder; or, the self-assembling peptide and the microspheres 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 microspheres are placed in another container in the form of a powder or solution; or, the self-assembling peptide is placed in one container in the form of a powder, and the microspheres are placed in another container in the form of a solution; or the peptide and the microspheres are placed in one container in the form of a solution.

[0119] In some embodiments, the self-assembling polypeptide and the initiator are placed in the same container in the form of powder, and the microspheres are placed in another container in the form of powder.

[0120] In some embodiments, the self-assembling polypeptide is placed in one container in the form of a hydrogel that forms a three-dimensional network scaffold structure in the presence of an initiator, and the microspheres are placed in another container in the form of a powder or solution.

[0121] 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 and under conditions where a wide range of endogenous substances exist in the human body, and can also perform the self-assembling peptide microsphere system of supporting microspheres and repairing functions in a solution state.

[0122] In a second aspect, the present invention provides a method for forming a microsphere filler from the composition of the first aspect, the method comprising: mixing the microspheres with self-assembling peptides, and inducing the self-assembling peptides to form a three-dimensional network scaffold material under conditions inducing a positive charge source.

[0123] In some embodiments, the method includes the steps of inducing the self-assembling peptides to form a three-dimensional network scaffold material under conditions of a positive charge source, and the step of adding microspheres to form a microsphere filler; or, the step of inducing the composition comprising the self-assembling peptides and microspheres to form a microsphere filler under conditions of a positive charge source.

[0124] In some embodiments, the method includes the step of initiating at a pH of less than 6.

[0125] When the pH of the self-assembling peptide solution is less than 6, it will self-initiate to form a hydrogel.

[0126] In some embodiments, the method includes the step of initiating with a positive charge source at a pH between 6-10.

[0127] In some embodiments, the method comprises the step of initiating with a positive charge source at pH 6-8.0, preferably pH 6.5-7.5, preferably pH 7.0-7.5, more preferably pH 7.2-7.4.

[0128] In some embodiments, the method comprises injecting a mixed solution of self-assembling peptides and microspheres into animal or human tissue at pH 6-8.0, preferably pH 6.5-7.5, preferably pH 7.0-7.5, and more preferably pH 7.2-7.4, to be triggered by the positive charge source substance therein.

[0129] The solution self-assembling peptide can be dissolved in a neutral or alkaline solvent, and the solution can be adjusted after dissolution. The solvent includes one or more aqueous solutions of sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonia water, etc. that can provide an alkaline environment.

[0130] The solvent can be used to dissolve the self-assembling peptides of the present invention as long as it can provide a neutral or alkaline environmental solution, preferably a physiologically acceptable solution. The pH of the mixed solution of the proteinaceous substance and the self-assembling peptide is adjusted to 6-10, preferably to 6.0-8.0, more preferably 6.5-7.5, and most preferably 7.0-7.5.

[0131] In some embodiments, the method comprises the following steps:

[0132] (1) dissolving the self-assembling peptide in a solvent;

[0133] (2) Add the microspheres to the polypeptide solution and mix evenly to obtain a microsphere filler.

[0134] In some embodiments, in step (1), the concentration of the self-assembling peptide is 0.5 wt% to 5.0 wt%.

[0135] In some embodiments, in step (1), the concentration of the self-assembling peptide after redissolution is 0.5 wt% to 5.0 wt%.

[0136] In some embodiments, in step (2), the concentration of the microspheres is 10 wt% to 40 wt%.

[0137] In some embodiments, the method comprises the following steps:

[0138] (1) dissolving the lyophilized powder of the self-assembling peptide in a solvent to form a self-assembling peptide solution;

[0139] (2) adding the microspheres to the self-assembling peptide solution obtained in step (1) to obtain a solution containing the self-assembling peptide and the microspheres.

[0140] After the solution containing self-assembling peptides and microspheres is injected into the tissue, it can self-assemble in response to proteins and other substances in the biological environment without introducing exogenous substances, thereby achieving medical aesthetic regeneration and filling.

[0141] Alternatively, in the presence of an initiator, the self-assembling peptide self-assembles to form a three-dimensional network structure, and then microspheres are added. The network structure formed by the self-assembling peptide supports the microspheres in a liquid state, so that the microspheres are evenly distributed in the peptide self-assembly network material, and a uniformly dispersed microsphere filler can be quickly obtained.

[0142] A convenient and safe method for preparing a broadly responsive, self-assembling peptide hydrogel under physiological conditions is suitable for use in laboratories, hospitals, and even the field. It is safe, highly operable, simple, and quick, requiring no adjustment of the system's pH, temperature, light, salt, or ion composition. The self-assembling peptide hydrogel of the present invention is formed in situ within half an hour, demonstrating strong operability. Gelation is initiated by endogenous substances, such as tissue fluids and other common biomedical substances, as well as some synthetic drugs. This reduces the requirements for the self-assembling peptide gelation process for applications such as tissue filling and repair, thereby better ensuring the biocompatibility of the self-assembling peptide hydrogel of the present invention.

[0143] The three-dimensional mesh scaffold material is a nanostructure.

[0144] The initiator is a positive charge source substance, and the positive charge source substance includes a substance with a positively charged group or a positively charged ion.

[0145] In some embodiments, the positive charge source substance is a biomolecule, drug, functional molecule, or small molecule such as metal ions, amino acids, etc., whose number of hydrogen bond acceptors is smaller than that of hydrogen bond donors, or a mixture comprising one or more of the above substances.

[0146] In some embodiments, the biomolecules include but are not limited to organic acids, proteins, polysaccharides and derivatives thereof, and the organic acids include but are not limited to lactic acid, tannic acid, citric acid and the like.

[0147] In some embodiments, the protein is selected from proteins that are capable of donating hydrogen ions under neutral physiological conditions.

[0148] Preferably, the proteins are independently selected from proteins having an isoelectric point (PI) value lower than 7.0 (preferably 3.4-6.05).

[0149] In some embodiments, the protein includes, but is not limited to, fibrinogen, globulin, hemoglobin, transferrin, laminin, fibronectin, vitronectin, and the like.

[0150] In some embodiments, the polysaccharide and its derivatives include but are not limited to chitin, chitosan, and the like.

[0151] In some embodiments, the drugs include but are not limited to antibiotics, dopamine, etc.; preferably, the antibiotics include but are not limited to kanamycin, gentamicin, etc.

[0152] In some embodiments, the functional molecules include but are not limited to antioxidants, cell proliferation promoting components, etc.;

[0153] In some embodiments, the antioxidants include but are not limited to vitamins such as niacinamide.

[0154] In some embodiments, the cell proliferation promoting component includes but is not limited to spermine, spermidine, and the like.

[0155] In some embodiments, the metal ions include, but are not limited to, potassium, calcium, magnesium ions, and the like.

[0156] In some embodiments, the amino acid includes but is not limited to amino acids or polymers thereof, such as lysine, arginine, polylysine, polyarginine, and the like.

[0157] In some embodiments, the positive charge source is urea or nicotinamide mononucleotide.

[0158] In some embodiments, the positive charge source substance is serum, plasma, animal or plant tissue fluid, or a mixture containing the above positive charge source substances.

[0159] It should be noted that if the hydrophobic domain is too hydrophobic, it will easily form an association, while if the hydrophobic domain is too weak, it will not be able to achieve self-assembly. The hydrophilic domain interacts with the initiator to trigger the self-assembly of the self-assembling peptide aqueous solution into a peptide hydrogel. Therefore, the selection of hydrophobic amino acids in the hydrophobic domain, the selection of hydrophilic amino acids in the hydrophilic domain, and the balance and precise combination of hydrophobic and hydrophilic amino acids are particularly important for the self-assembling peptides of the present invention to not form a hydrogel under neutral conditions and to form a hydrogel under initiation conditions.

[0160] The present invention unexpectedly discovered that self-assembling peptides with the described structure can initiate and self-assemble into nanonetwork structures under physiological conditions, as well as under the presence of a wide range of endogenous substances in the human body. In particular, the use of endogenous physiological substances, such as serum, plasma, and animal and plant tissue fluids, as initiators significantly reduces safety risks in clinical applications, which is particularly important for drug delivery, human tissue repair, and regenerative medicine.

[0161] In some embodiments, the microsphere filler is in the form of a hydrogel or in a dried form of a hydrogel, such as in the form of a lyophilized powder of a hydrogel.

[0162] In a third aspect, the present invention provides a microsphere filler comprising the composition of the first aspect, or prepared by the method of the second aspect.

[0163] In some embodiments, the microsphere filler is in the form of a hydrogel or in a dried form of a hydrogel, such as in the form of a lyophilized powder of a hydrogel.

[0164] In some embodiments, the microsphere filler is a polypeptide solution or polypeptide powder.

[0165] In some embodiments, the microsphere filler is in the form of an injectable hydrogel.

[0166] In some embodiments, the microsphere filler is a nanostructure.

[0167] The three-dimensional network scaffold material has more β-sheet structures than the self-assembling peptide.

[0168] In a fourth aspect, the present invention provides the use of the composition of the first aspect, the method of the second aspect, and the microsphere filler of the third aspect in one or more of the following: regenerative medicine and tissue regeneration; drug delivery; wound healing; implantable materials; gene therapy; stem cell therapy; and medical cosmetology. DETAILED DESCRIPTION

[0169] The present invention obtains a self-assembling peptide by cleverly controlling the secondary structure of the polypeptide, uniquely selecting hydrophobic amino acids in the hydrophobic domain, uniquely selecting hydrophilic amino acids in the hydrophilic domain, and balancing and precisely coordinating the hydrophobic and hydrophilic amino acids.

[0170] The self-assembling peptide responds to common positive ion / group source substances in the body under neutral conditions of human physiological conditions to form a three-dimensional nanofiber network structure containing the self-assembling peptide.

[0171] The self-assembling peptides of the present invention are in a neutral liquid state during use under human physiological conditions, thus avoiding the risks caused by adjusting pH or introducing other exogenous substances, such as certain metal salt ions, specific proteins, etc.

[0172] The self-assembling peptides of the present invention have a stronger support effect due to the presence of hydroxyproline (O) in the β-turn motif of the hydrophilic domain. In addition, the present invention has found that the presence of hydroxyproline (O) compared to proline (P) in the β-turn motif of the hydrophilic domain, the support effect of the self-assembling peptide is stronger.

[0173] The self-assembling peptide can self-initiate to form a hydrogel in a solution when the pH value is less than 6.

[0174] When the pH of the self-assembling peptide is between 6 and 10, a positive charge source substance is added as an initiator to form a hydrogel.

[0175] The interaction between the acidic amino acids of the self-assembling peptides of the present invention and the initiator triggers the self-assembly of the peptide aqueous solution into a peptide hydrogel. Under neutral conditions, the addition of the initiator produces a positively charged substance, which neutralizes the negatively charged acid ions on the polypeptide molecules, thereby reducing the repulsive forces between the polypeptide molecules. The polypeptide molecules then self-assemble through hydrophobic interactions and hydrogen bonds, ultimately forming a nanoscale three-dimensional network structure.

[0176] The present invention provides a rapidly resolvable polypeptide microsphere filler. The self-assembling peptide molecules in the formulation form a complex in aqueous solution, providing dispersion and support for the PLLA microspheres. Furthermore, upon injection of the formulation into tissue, the self-assembling peptides rapidly self-assemble into a hydrogel with a three-dimensional network-like scaffold structure, responding to specific proteins in the tissue. This hydrogel provides immediate cosmetic benefits and creates a regenerative microenvironment. The proteins that can trigger polypeptide self-assembly include laminin, fibronectin, fibrinogen, transferrin, gamma globulin, vitronectin, and hemoglobin. Laminin, fibronectin, and vitronectin are important components of the natural extracellular matrix, allowing the formulation to rapidly gel upon entry into tissue. Fibrinogen, transferrin, gamma globulin, and hemoglobin are proteins widely present in serum or plasma, making the formulation potentially valuable for wound coagulation and repair, as well as for the normal immune function of organisms.

[0177] The self-assembling peptide solution in the preparation of the present invention not only has the advantage of quickly assembling to form a hydrogel in a short period of time, but also has the characteristics of shear thinning and rapid recovery to the original state after the external force is removed. This shows that under the action of strong external mechanical force, some non-covalent bonds inside the self-assembling peptide hydrogel will break, and the macroscopic manifestation is that the gel state is destroyed and becomes a solution; when this mechanical force disappears, these broken non-covalent bonds will reconnect, and the macroscopic manifestation is that the destroyed gel recovers to a state similar to the original gel through self-repair. In addition, the recovery time of the hydrogel does not exceed 10 minutes, and the storage modulus after recovery is at least 70% of the original, and can even reach more than 85% of the original, or even more than 95%. Furthermore, the self-assembling peptide hydrogel of the present invention still has the ability to self-repair after undergoing multiple shear thinning, that is, shear thinning can only temporarily destroy the three-dimensional network structure inside the peptide hydrogel, and has the potential to form gel in situ after injection. Shear thinning can be achieved by using a variety of mechanical forces that can exert shear or shear stress on the self-assembling peptide hydrogel, such as vibration, injection, etc. That is, after the microsphere filler is injected into the tissue, it can still be extracted by syringe and then injected or vibrated to achieve further shaping changes.

[0178] The microsphere filler of the present invention performs its dispersing function without the aid of other ingredients. Once injected into tissue, it self-assembles in response to proteins in the biological environment without introducing exogenous substances, thereby achieving aesthetic regeneration and filling. Furthermore, the microsphere filler of the present invention does not alter the temperature and pH of the entire system during the self-assembly process and / or after gel formation, significantly enhancing its safety in clinical aesthetic applications.

[0179] The three-dimensional network structure formed by non-covalent bonds exhibits self-healing properties. When the medical aesthetic microspheres are added and shaken, they disrupt the non-covalent bonds within the three-dimensional network, achieving uniform dispersion. Within tens of nanoseconds, the three-dimensional network automatically repairs itself through non-covalent forces, securing the medical aesthetic microspheres within the network structure and maintaining their uniform dispersion within the self-assembling peptide solution. Therefore, after adding the medical aesthetic microspheres, a gentle shake will evenly disperse them within the self-assembling peptide solution, resulting in a simple finished medical aesthetic microsphere filler. Furthermore, the self-assembling peptide can support the microspheres in a liquid state. Since the system can directly initiate gelation upon entry into the human body, the peptide achieves an immediate cosmetic effect, while the microspheres provide a long-lasting cosmetic effect. The long-term and short-term cosmetic effects are combined through the peptide's self-assembly behavior.

[0180] The gel formed by the self-assembling peptides has a very large surface area, allowing nutrients, bioactive molecules, and oxygen to diffuse fully. At the same time, the fibrous mesh scaffold material formed by the self-assembling peptides is similar to the extracellular matrix, providing cells with a nanoscale microenvironment that is conducive to cell adhesion and growth. Therefore, while the self-assembling peptides play a filling role, they fully promote the proliferation and differentiation of the body's cells within the self-assembling peptide material. During the degradation of the self-assembling peptide material, the body's cells continue to proliferate and differentiate in the self-assembling peptide scaffold, forming normally developed body tissues at the filling site. Even if the self-assembling peptides are completely degraded, the body tissues formed at the filling site will continue to grow normally, achieving a "permanent" filling effect and solving the problem of the filling effect disappearing as the filler material degrades.

[0181] Compared to the mixed gel obtained by mixing hyaluronic acid and microspheres, which occasionally exhibits uneven dispersion and clumping, leading to injection difficulty and pain, the mixed gel obtained using the self-assembling peptides of the present invention, due to the small molecular weight of the polypeptides and the fact that they assemble into nanofibers through non-covalent bonds, the microspheres within the system are individually wrapped in fine and dense polypeptide fibers, fundamentally avoiding the phenomenon of microspheres agglomerating into clumps, which causes injection difficulty and pain. Furthermore, the use of self-assembling peptides as fillers eliminates the need for chemical modification of the microspheres and various fillers, reducing production costs.

[0182] Hyaluronic acid injections can cause unexpected problems such as clumping, facial stiffness, foreign body reactions, and allergic reactions if the injection volume is excessive, a large hyaluronic acid molecule is used, the physician is inadequately skilled, or the patient is unsuitable for hyaluronic acid injections. Self-assembling peptide hydrogels are formed through non-covalent bonds, and their relatively flexible mechanical properties prevent post-injection clumping, stiffness, and foreign body reactions. Furthermore, as an ideal tissue engineering scaffold material, self-assembling peptide hydrogels can provide a microenvironment similar to the natural extracellular matrix for cell proliferation and differentiation. They are characterized by high purity, simple composition, biodegradability, lack of immune response, lack of animal-derived contamination, and amino acid degradation products in the body. They have been successfully applied in cell engineering, tissue engineering, tissue repair, and controlled drug release. Using self-assembling peptides as fillers can completely avoid allergic reactions.

[0183] Compared to conventional hyaluronic acid fillers, which metabolize into CO2 and water within 1-2 days, cross-linking (i.e., cross-linking hyaluronic acid) maintains its effectiveness for approximately 6-9 months, with some products maintaining efficacy for 1-2 years. In contrast, the hydrogel formed by self-assembling peptides has a very large surface area, allowing for ample diffusion of nutrients, bioactive molecules, and oxygen. Furthermore, the fibrous mesh scaffold material formed by the self-assembling peptides resembles the extracellular matrix, providing a nanoscale microenvironment for cells to adhere and grow. Therefore, while performing the filling function, the self-assembling peptides also fully promote the proliferation and differentiation of cells within the self-assembling peptide material. During the degradation of the peptide material, the body's cells continue to proliferate and differentiate within the self-assembling peptide scaffold, forming normally developed tissues at the filled site. Even after the self-assembling peptides are completely degraded, the tissue formed at the filled site continues to grow normally, achieving a "permanent" filling effect and resolving the problem of the filling effect disappearing with the degradation of the filler material. Therefore, the self-assembling peptide can play the role of uniformly dispersing PLLA microspheres in the composite preparation, and the hydrogel formed by its self-assembly can construct a regenerative environment in clinical applications, so that the microsphere filler can play a perfect regenerative filling role.

[0184] Glossary:

[0185] 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.

[0186] Hydrogels have the following properties:

[0187] 1. Good biocompatibility: The polymer contains a large number of hydrophilic groups, which can absorb dozens of times more water than its own amount, and has the characteristics of swelling but not dissolving in water, and has good water retention capacity;

[0188] 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;

[0189] 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.

[0190] 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.

[0191] Peptide Self Assembly is a short chain of amino acids with alternating charge and polarity domains. When dissolved in a neutral solvent and physiological salt concentration, these peptides spontaneously assemble into hierarchical nanostructures through hydrogen bonds, ionic bonds, hydrophobic interactions, or van der Waals forces. Materials derived from these assemblies have the advantages of being non-toxic, non-immunogenic, non-thrombogenic, degradable, and easily metabolized. At the same time, nanofibers have the same size scale as natural ECM fibers and can be easily designed to mimic the stiffness of various soft tissues. They can also be further functionalized by attaching cell-interacting peptide domains or cytokines and growth factors. Therefore, they can be used to design biologically relevant culture environments and improve the control of proliferating cell populations.

[0192] Self-assembling peptides can be used as drug carriers in hydrogels for wound treatment and as peptide nanofibers for cancer treatment, enabling the sustained release of small molecules, growth factors, and monoclonal antibodies. For example, self-assembling peptides can be used to promote angiogenesis within regenerating tissues and to repair skin wounds using peptide-based scaffolds. However, the application of self-assembling peptides in injectable therapeutic hydrogels is still in its infancy.

[0193] 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.

[0194] "Amino acid" includes those naturally occurring as well as non-naturally occurring amino acids, such as D-natural amino acids, beta and gamma derivatives. According to standard terminology, amino acid residue sequences can be named using three-letter or one-letter codes, for example: alanine (Ala, A); arginine (Arg, R); asparagine (Asp, N); aspartic acid (Aspartic acid, D), cysteine ​​(Cysteine, C); glutamine (Glutamine, Q); glutamic acid (Glutamic acid, E); glycine (Gly, G); histidine (His, H), isoleucine (Ile, I); leucine (Leu, L), lysine (Lysine, L), methionine (Met, M); phenylalanine (Ala, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W), tyrosine (Tyr, Y); valine (Val, V); selenocysteine ​​(Sec, U); hydroxyproline (Hyp, O).

[0195] In the present invention, a "peptide" is an amino acid chain. In particular, a peptide is 2 to 40 amino acids in length.

[0196] In the present invention, "self-assembly" refers to the aggregation of polypeptides into an ordered structure under normal environmental conditions.

[0197] 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.

[0198] 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.

[0199] The term "hydrophobicity" used in the present invention refers to a property of being inclined to repel water or being completely insoluble in water.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] "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.

[0204] When the protein solution is at a certain pH, the protein has an equal tendency to dissociate into positive and negative ions, that is, it becomes a zwitterionic ion with a net charge of zero. The pH of the solution at this time is called the isoelectric point (pI) of the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0205] Figure 1A shows that the self-assembling peptide is in liquid form when arginine is used as an initiator and no initiator is added. Figure 1B shows that when arginine is present, the self-assembling peptide is a hydrogel. Figure 1C shows that when tissue fluid is used as an initiator, the self-assembling peptide can form a hydrogel, which remains in a gel state after being squeezed out of a syringe.

[0206] 2A-2J show that different initiating substances mixed with self-assembling peptides can all produce the three-dimensional network scaffold material of the present invention.

[0207] FIG3 shows a hydrogel three-dimensional mesh scaffold material formed using fibrinogen, transferrin, and γ-globulin as initiators.

[0208] 4A-4B show the fluorescence changes of thioflavin T in peptide fiber hydrogels formed using fibrinogen, transferrin, and γ-globulin as initiators.

[0209] FIG5 shows the effects of different sequence structures of self-assembling peptides on the support of red blood cells in forming a three-dimensional mesh scaffold.

[0210] Figure 6 shows the changes in rheological properties of different self-assembling peptide sequence structures, which are SEQ ID NO: 7 (A), SEQ ID NO: 3 (B), SEQ ID NO: 2 (C), SEQ ID NO: 1 (D), SEQ ID NO: 5 (E), SEQ ID NO: 6 (F), SEQ ID NO: 4 (G), SEQ ID NO: 10 (H), and SEQ ID NO: 8 (I).

[0211] FIG7 shows the modulus results of a solution of a mixture of the protein-responsive self-assembling peptide of SEQ ID NO: 14 and protein after 30 minutes, wherein the concentration of the protein-responsive self-assembling peptide is 1 wt %.

[0212] FIG8 shows the modulus results of a solution of a mixture of the protein-responsive self-assembling peptide of SEQ ID NO: 18 and protein after 30 minutes, wherein the concentration of the protein-responsive self-assembling peptide is 1 wt %.

[0213] FIG9 shows a graph of shear thinning and recovery experimental results of the hydrogel of the present invention.

[0214] FIG10 shows a confocal laser scanning electron microscopy image of a hydrogel formed by the protein of the present invention and used to support cells, wherein the concentration of the self-assembling peptides is 0.1 wt %.

[0215] FIG11 shows the effects of not (2D) and using (3D) the self-assembling peptide scaffold solution on the cell survival of mouse mesenchymal stem cells during refrigerated storage.

[0216] Figure 12 shows that 0.3wt.%, 0.5wt.%, and 0.7wt.% of the self-assembling peptide of the present invention were added to the cell culture system to induce self-assembly into a fiber network scaffold and culture liver cancer cells. The cell sphere diameters were counted on the third day, seventh day, eleventh day, and fifteenth day, and the characteristic cell spheres in the culture system were photographed.

[0217] Figure 13 shows that the addition of 0.3wt.% of the self-assembling peptide of the present invention to the cell culture system triggered its self-assembly into a fiber network scaffold and cultured porcine muscle satellite cells. The cell sphere diameters were counted on the first, second, third and fourth days, and the characteristic cell spheres on the first, second and fourth days in the culture system were photographed.

[0218] Figure 14 shows that the self-assembling peptide solution can stably disperse L-polylactic acid microspheres: among them, A shows that the microspheres precipitate in the aqueous solution, but are evenly suspended and dispersed after mixing with the self-assembling peptide solution; B shows that the self-assembling peptide mixture of the microspheres is liquid; C shows that after the self-assembling peptide mixture of the microspheres obtained in Figure 14B is mixed with tissue fluid, the self-assembling peptide / L-polylactic acid microsphere solution quickly forms a hydrogel.

[0219] Figure 15 shows that the self-assembling peptide solution can stably disperse polycaprolactone: among them, A shows that the microspheres precipitate in the aqueous solution, but are evenly suspended and dispersed after mixing with the self-assembling peptide solution; B shows that the self-assembling peptide mixture of the microspheres is liquid; C shows that after the self-assembling peptide mixture of the microspheres obtained in Figure 15B is mixed with tissue fluid, the self-assembling peptide / L-polylactic acid microsphere solution quickly forms a hydrogel.

[0220] The present invention is further explained below through specific examples. Since the principle of the self-assembling peptides involved in the present invention to form hydrogels in response to different proteins is consistent, in multiple embodiments, different self-assembling peptide sequences are used as examples to provide verification data on 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 peptides of the present invention, for which detailed data are not provided in the present invention, also have equivalent or similar effects. The amino acids mentioned in the present invention are characterized by single-letter abbreviations, which are generally accepted in the art.

[0221] Example

[0222] Example 1. Synthesis of self-assembling peptide compounds

[0223] The self-assembling peptides or peptide-like compounds of the present invention are synthesized by a standard solid-phase self-assembling peptide synthesis method, and the amino acid sequences are shown in SEQ ID NOs: 1-32.

[0224] Example 2. Preparation of Polar Peptide or Peptoid Solution

[0225] The self-assembling peptide or peptoid of Example 1 is added to water or phosphate buffer (PBS) buffer (pH = 7.2-7.4, unless otherwise specified, the PBS buffer used in this description is of this pH value), and alkali solution is added dropwise until the self-assembling peptide is completely dissolved. The solution is adjusted to a neutral pH of 7.2 to obtain a 5 wt.% self-assembling peptide or peptoid mother solution, which is sterilized by autoclaving and stored at 4°C for future use. The mother solution is used to adjust the concentration of the self-assembling peptide to obtain a self-assembling peptide or peptoid material of a predetermined concentration. A certain amount of the self-assembling peptide or peptoid mother solution is diluted with PBS buffer to obtain a 0.5 wt.% self-assembling peptide or peptoid solution.

[0226] Example 3. Preparation of three-dimensional network scaffold materials formed by self-assembling peptides under the stimulation of positive ions / groups

[0227] The self-assembling peptide or peptoid solution obtained in Example 2 is evenly mixed with the initiating substance solution, and the concentration ratio of the self-assembling peptide or peptoid to the initiating substance in the mixed solution is ensured to be in the range of (1-100):(1-100), thereby obtaining a self-assembling, three-dimensional network scaffold with multiple initiating substances. The pH value of the obtained mixed solution is neutral (about pH 6 to about pH 8, preferably about pH 6.5-7.5, preferably about pH 7-7.5).

[0228] Taking the self-assembling peptide represented by the amino acid sequence IIIIIGSIIGPGGDGPGGV (SEQ ID NO: 1) as an example, the initiating substance is arginine. A 0.5 wt.% peptide solution (see FIG1A ) is in liquid form. When the sample bottle is inverted, the solution flows back and collects at the bottle mouth. In the presence of arginine, a peptide hydrogel with a peptide concentration of 0.5 wt.% self-assembles in response to the initiating substance arginine (see FIG1B ). When the sample bottle is inverted, the hydrogel does not fall. Using tissue fluid as the initiator, the self-assembling peptide can form a hydrogel that remains in a gel state after being extruded from a syringe (see FIG1C ). This also has the same or similar effects on peptide hydrogel materials formed in response to other types of initiating substances.

[0229] Taking the self-assembling peptide represented by the amino acid sequence LLLLLGSVLGPAGEGPAGE (SEQ ID NO: 29) as an example, a 2% concentration of the self-assembling peptide quickly forms a hydrogel after being mixed with an equal volume of human tissue fluid. The hydrogel can be drawn up with a syringe and remains in a gel state after being extruded by injection. It also has the same or similar effects on peptide hydrogel materials formed in response to other types of triggering substances.

[0230] According to this method, a self-assembling peptide hydrogel material with any predetermined peptide concentration ratio of the self-assembling peptide or peptoid to the initiating substance described in the present invention can be prepared. The results are shown in Table 1 below.

[0231] Example 4: Structural Characterization of the Scaffold Material Formed by the Self-Assembling Peptides of the Present Invention

[0232] Experiment 1: Transmission Electron Microscopy (TEM)

[0233] The polar peptide or peptoid is exemplified by SEQ ID NO: 3 (IIIIIGSIIGOGGEGPGGV); the initiating substance is exemplified by polylysine, spermine, gentamicin, lysine, arginine, spermidine, kanamycin, chitosan, and magnesium ions.

[0234] Experimental method: The mother liquor of the self-assembling peptide material obtained in Example 3 (SEQ ID NO: 3) and the three-dimensional mesh scaffold material formed by self-assembly in response to the initiating substance obtained in Example 2 were diluted with ultrapure water. In order to obtain the nanoscale morphology of the hydrogel under physiological conditions, they were incubated at 37°C for 1 hour, 10 μL was taken and placed on a 300-mesh carbon support film copper grid (Xinxing Bairui), and vacuum dried. The sample was stained with a 2wt.% phosphotungstic acid negative staining solution for 60 seconds each time, repeated three times. The stained copper grid was placed at room temperature to dry. Imaging observation was performed using a Talos G2 200X transmission electron microscope.

[0235] Experimental results: As shown in Figures 2A-2J, different initiating substances can be mixed with self-assembling peptides to obtain the three-dimensional mesh scaffold material of the present invention. The initiating substances corresponding to AJ are: polylysine, spermine, gentamicin, lysine, arginine, spermidine, kanamycin, chitosan, and magnesium ions. The structure formed by the self-assembling peptide material (Figure 2A) is composed of curved and interwoven nanofibers. The fibers are unevenly distributed, curved, and highly flexible. The presence of the initiating substance reduces the curvature of the fibers (Figures 2B-2J), significantly increases the fiber length and diameter, and changes the arrangement of the fibers. The fibers are tightly and orderly arranged to form fiber bundles, which are interwoven with each other.

[0236] Experiment 2: Circular Dichroism (CD) Analysis

[0237] Circular dichroism spectroscopy is the most widely used method for determining protein secondary structure and monitoring conformational changes of protein molecules induced by external conditions. This method is used to detect liquids, and the results obtained are closer to the secondary structure of proteins in real physiological environments. It is a fast, simple and relatively accurate method for studying protein conformation.

[0238] Experimental method: Preparation of self-assembling peptide or derivative solution for CD detection: The obtained 0.2 wt % self-assembling peptide solution was diluted with PBS buffer to a self-assembling peptide concentration of 0.02 wt %.

[0239] Preparation of protein solution for CD detection: Laminin, fibronectin, fibrinogen, transferrin, γ-globulin, and vitronectin were added to ultrapure water to a protein concentration of 0.02 wt%.

[0240] Preparation of self-assembling peptide or derivative samples: The 0.2 wt % self-assembling peptide solution obtained in Example 2 was diluted with PBS buffer to a self-assembling peptide concentration of 0.01 wt %, and incubated at 37° C. for one hour.

[0241] To prepare the peptide hydrogel samples of the present invention, a 0.02 wt% self-assembling peptide solution was mixed with a 0.02 wt% protein solution at a 1:1 volume ratio. The mixture was incubated at 37°C for one hour to obtain a final self-assembling peptide concentration of 0.01 wt%. Because the self-assembling peptide / protein hydrogel is a swelling system, although the self-assembling peptide concentration in this experiment was lower than 0.1 wt%, the results still confirmed the interaction between the self-assembling peptide and the protein.

[0242] 400 μL of each prepared sample was placed in a rectangular quartz cell with a 1 mm optical path length. Circular dichroism detection was performed in the 190 nm to 260 nm wavelength range using a MOS-450 / AF-CD spectrometer (Bio-Logic, Claix, France) at room temperature with a resolution of 0.5 nm and a scan speed of 0.5 nm / s. Each set of measurements was performed in triplicate after background subtraction. The results are shown in Figure 3 for peptide hydrogels formed with the self-assembling peptide of SEQ ID NO: 13 (IIIIIGOGIIGPGGEGPGGE) and fibrinogen, transferrin, and gamma globulin, respectively.

[0243] The results of circular dichroism detection show that the responsiveness of the self-assembling peptide or its derivatives to proteins is manifested as a change in secondary structure. Figure 3 shows that when the self-assembling peptide or its derivatives exist alone (see control group) in a neutral environment, the secondary structure of the nanofibers assembled therein is mainly β-turns. In the system where proteins are introduced separately, it can be seen that the positive peak near 200nm in the circular dichroism spectrum shifts to varying degrees. This shows that compared to when the self-assembling peptide or its derivatives exist alone and are not triggered, the response to protein increases the secondary structure of the nanofibers assembled by the self-assembling peptide or its derivatives, providing a favorable basis for forming more β-fold structures and making the fiber structure more regular and orderly. In addition, a negative peak appears near 192nm when the protein group is added (see fibrinogen triggering group, transferrin triggering group, and gamma globulin triggering group). This is the embodiment of the characteristic polymeric structure formed by the participation of proline or hydroxyproline in the self-assembling peptide in the assembly of the self-assembling peptide in the fiber secondary structure. This shows that the response to these three substances makes the secondary structure of the fiber assembled by the self-assembling peptide of the present invention more diverse, and the increase in secondary structure promotes the stability of the fiber structure. This experiment shows that although self-assembling peptides or their derivatives can assemble into nanofibers with specific secondary structures by themselves, their responsiveness to proteins enables them to assemble into nanofibers with more complex and ordered structures, thus macroscopically exhibiting a three-dimensional network structure with tight cross-linking and uniform pores as shown in Experiment 1.

[0244] Experimental Conclusion: Experiment 2 demonstrated that the presence of the initiating substance caused the polar peptides to self-assemble into a gel-like network, which is beneficial for cell adhesion and three-dimensional culture. Combining Experiments 1 and 2, it was confirmed that the interaction between the self-assembling peptides and the initiating substance altered the self-assembly pathway, promoting the formation of a three-dimensional network scaffold material with a distinct microscopic morphology compared to that observed without the initiating substance.

[0245] When the self-assembling peptide or peptoid of the present invention is used, or when the self-assembling peptide structure of the present invention is met, similar experimental results to those of Experiments 1 and 2 can be obtained under the stimulation of the triggering substance, and are not listed in detail one by one.

[0246] Experiment 3: Thioflavin T fluorescence experiment

[0247] Experimental Methods: Thioflavin T can bind to the β-sheet structure of self-assembling peptides, thereby enhancing fluorescence intensity. In this experiment, we used SEQ ID NO:13 (IIIIIGOGIIGPGGEGPGGE) to monitor the fluorescence changes of Thioflavin T and verify the secondary structural changes of self-assembling peptides during self-assembly. 800μM (0.148 wt%), 400μM (0.072 wt%), and 200μM (0.036 wt%) peptide sols were mixed with 0.072 wt% of a trigger in a 1:1 volume ratio. Ultrapure water was used instead of the trigger for the control. After 15 minutes of stabilization, the sols were mixed with an equal volume of a 100μM Thioflavin T solution. Fluorescence emission was measured in the 450-550 nm range, with five spectra collected for each sample. The excitation wavelength was 442 nm, and the excitation and emission slits were 5 nm and 2.5 nm, respectively.

[0248] Experimental Results: CD spectral data were validated using thioflavin T (ThT), which can stain peptide fibers rich in β-sheet structure, confirming the presence of β-sheets and revealing their relationship with peptide concentration. β-sheets were present in the self-assembling peptide sol, and their content was positively correlated with the self-assembling peptide concentration, indicating that the self-assembling peptide is concentration-dependent (Figure 4A). This is because the increase in peptide monomers facilitates intermolecular contact, thereby increasing the degree of assembly. After adding the initiator, the β-sheet content of the gel-generated group was much higher than that of the sol group (Figure 4B). CD and ThT staining data indicate that under neutral pH conditions, the supramolecular polymers generated by protein initiator-activated self-assembling peptides exhibit a typical β-sheet structure.

[0249] 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

[0250] Experiment 1: Verification of red blood cell support by different sequences under liquid conditions

[0251] In this experiment, taking red blood cells as an example, we verified the supporting effect of amino acid sequences on cells, especially the supporting effect of hydroxyproline and alanine in the amino acid sequence on cells.

[0252] Experimental method: SEQ ID NO: 1 (IIIIIGSIIGPGGDGPGGV), SEQ ID NO: 2 (IIIIIGSIIGPGGEGPGGV), SEQ ID NO: 3 (IIIIIIGSIIGOGGEGPGGV), and SEQ ID NO: 7 (IIIIIIGSIIGOGAEGPGGV) self-assembling peptide stock solutions were prepared with PBS buffer at a concentration of 0.1 wt%, and sterilized at high temperature.

[0253] In a 2ml clear glass vial, 2mL of self-assembling peptide solution (containing fibronectin at a final concentration of 0.1wt%) was prepared for each of the four materials at 0.1wt%, 0.05wt%, and 0.01wt%, respectively. A blank control (containing 0.1wt% fibronectin but no self-assembling peptide solution) was also prepared. 5×109 red blood cell stock solution was added to a final concentration of 1×108 and the mixture was pipetted evenly. Images were taken every 4 hours to compare the red blood cell support effects of the different materials.

[0254] Experimental conclusion: Among the self-assembling peptides with similar amino acid sequences, hydroxyproline and alanine have a significant effect on the cell support ability of the scaffold solution under liquid conditions. As shown in Figure 5, under high concentration conditions (0.1wt%, 0.05wt%), the four self-assembling peptide solutions can effectively support the cell support of 1×10 8Red blood cells; however, under low concentration conditions (0.01 wt%), hydroxyproline, alanine, and glutamic acid in the hydrophilic domain showed a significant impact on the supporting ability of red blood cells. Obvious sedimentation occurred in the four self-assembled peptide solutions at 8 hours, 24 hours, 32 hours, and 48 hours respectively. The supporting ability of the four self-assembled peptides from strong to weak is: SEQ ID NO:1 (IIIIIGSIIGPGGDGPGGV) < SEQ ID NO:2 (IIIIIGSIIGPGGEGPGGV) < SEQ ID NO:3 (IIIIIGSIIGOGGEGPGGV) < SEQ ID NO: 7 (IIIIIGSIIGOGAEGPGGV). Based on the scaffold network formed in response to fibronectin, compared with SEQ ID NO:1, glutamic acid in the hydrophilic region of SEQ ID NO:2, glutamic acid and hydroxyproline in the hydrophilic region of SEQ ID NO:3, and glutamic acid, hydroxyproline, and glycine in the hydrophilic region of SEQ ID NO:7 successively showed an increasingly stronger enhancing effect on cell supporting ability. Hydroxyproline is abundantly present in the collagen tissue of animals and can enhance the elasticity and supporting force of the protein matrix through hydrogen bonds formed by its hydroxyl groups. In this patent, by introducing hydroxyproline, the stability of the β-sheet formed by the self-assembly of self-assembled peptides can be significantly enhanced through the enhanced hydrogen bond network between self-assembled peptides, thereby improving the stability of the self-assembled scaffold and enhancing the cell supporting function. At the same time, through the substitution of glycine by single alanine and the substitution of aspartic acid by glutamic acid in the β-sheet structure in the hydrophilic region, the hydrophobicity of the amino acid side chains can be enhanced without destroying the β-turn structure in the hydrophilic region, and the stability of the self-assembled material can also be increased through hydrophobic interactions, making the scaffold material more supportive of cells.

[0255] Experiment 2, Rheological Experiment, Influence of the Secondary Structure and Amino Acid Sequence of Self-Assembled Peptides

[0256] Experimental method: Preparation of self-assembling peptide material samples: Use the 2 wt% self-assembling peptide solution obtained in Example 2, SEQ ID NO: 1 (IIIIIGSIIGPGGDGPGGV), SEQ ID NO: 2 (IIIIIGSIIGPGGEGPGGV), SEQ ID NO: 3 (IIIIIIGSIIGOGGEGPGGV), SEQ ID NO: 4 (IIIIGSIIGOGGEGPGGV), SEQ ID NO: 5 (IIIIIGSIIGOGGEGPGGGV), SEQ ID NO: 6 (IIIIIGSIIGOGGEGPGGV), SEQ ID NO: 7 (IIIIIGSIIGOGAEGPGGV), SEQ ID NO: 8 (IIIIIGSIIOGGAEGPGGV), SEQ ID NO: 9 (IIIIIIGSIIGOGGVGPGGV), SEQ ID NO: 10 (IIIIIIGSIIGOGAEGPGGVGPG GV).

[0257] Preparation of hydrogel samples formed by self-assembling peptide solution in response to complete cell culture medium: dilute the 2 wt% self-assembling peptide material mother solution obtained in Example 2 with phosphate buffer, add calf serum, and the final concentration of self-assembling peptide is 0.5%, and the final concentration of serum is 10 v / v%, and the pH is neutral.

[0258] The storage modulus (G') of the mixed solution was measured using a MARS 60 rheometer on a 20 mm plate. To determine the rate of 3D nanomatrix formation, the solution was placed on the plate immediately after preparation. A 500 μm gap was used, with mineral oil added to prevent sample dehydration, and data collection began. A dynamic time sweep (DTS) experiment was performed to monitor the change in storage modulus (G') over time (1 Hz frequency, 1% strain) for 2000 minutes.

[0259] Experimental results: The mechanical strength of the peptide hydrogel of the present invention can effectively provide support and encapsulation effects for cells, functional molecules, drugs, etc., which is conducive to the realization of three-dimensional cell culture, tissue repair, drug sustained release, etc. Experiments have shown that by mixing the peptide solution and serum, self-assembling peptides can be immediately obtained to respond to the triggering substance and assemble into a hydrogel material with a storage modulus greater than 10. The operation is convenient and the gelation time is short. Comparing different sequences, it was found (see Figure 6) that SEQ ID NO:7 (IIIIIGSIIGOGAEGPGGV)>SEQ ID NO:3 (IIIIIGSIIGOGGEGPGV)>SEQ ID NO:2 (IIIIIGSIIGPGGEGPGGV)≥SEQ ID NO:1 (IIIIIIGSIIGPGGDGPGGV); this experimental phenomenon is consistent with the results of Experiment 3, that is, the replacement of glycine (G) with alanine (A) in hydroxyproline and hydrophilic regions can enhance the mechanical strength of the hydrogel formed by the present invention, thereby enhancing the support function of the scaffold material. At the same time, the results of comparing SEQ ID NO: 5 (IIIIIGSIIGOGGEGPGGGV) with SEQ ID NO: 6 (IIIIIGSIIGOGGEGPGV) showed that when the number of β-turn amino acids was 6 (SEQ ID NO: 5) and 3 (SEQ ID NO: 6), and the β-turn structure was increased to 3 consecutive (SEQ ID NO: 10), or the number of hydrophobic amino acids was 4 (SEQ ID NO: 4), the self-assembling peptide could still form a hydrogel structure with weak elasticity. In Figure 6, A corresponds to the sequence SEQ ID NO: 7, B corresponds to the sequence SEQ ID NO: 3, C corresponds to the sequence SEQ ID NO: 2, D corresponds to the sequence SEQ ID NO: 1, E corresponds to the sequence SEQ ID NO: 5, F corresponds to the sequence SEQ ID NO: 6, G corresponds to the sequence SEQ ID NO: 4, H corresponds to the sequence SEQ ID NO: 10, and I corresponds to the sequence SEQ ID NO: 8.

[0260] In SEQ ID NO:8, we completely destroyed the formation conditions of the first β-turn in SEQ ID NO:7 by adjusting the 10th-11th amino acid GO in SEQ ID NO:7 to OG, forming the sequence SEQ ID NO:8; As a result, we found that SEQ ID NO:8 completely lost its self-assembly response to serum. This phenomenon further proves that two consecutive β-turns are the most basic conditions for the self-assembly ability of the self-assembling peptide in the present invention. By comparing SEQ ID NO:9 (IIIIIGSIIGOGGVGPGGV) with SEQ ID NO:3 (IIIIIGSIIGOGGEGPGGV), we found that when the end of the β-turn is no longer connected to an acidic amino acid, the self-assembling peptide aqueous solution no longer responds to serum, and it can form a hydrogel in a neutral aqueous solution; here we further verified that the acidic amino acid connected to the end of the β-turn in the hydrophilic region of at least one self-assembling peptide is a prerequisite for the self-assembling peptide self-assembly material of the present invention to respond to the triggering component.

[0261] Example 6: Determination of hydrogel mechanical properties

[0262] Experiment 1: Determination of mechanical strength of hydrogel

[0263] 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 %.

[0264] Preparation of protein solutions for dynamic rheological testing: Use ultrapure water to prepare laminin solution, fibronectin solution, fibrinogen solution, vitronectin solution, γ-globulin solution, transferrin solution, hemoglobin solution, and human collagen solution with a protein concentration of 2 wt%.

[0265] Preparation of self-assembling peptide samples: The 2 wt % self-assembling peptide stock solution of Example 2 was diluted to 1 wt % with ultrapure water.

[0266] Preparation of hydrogel samples: 2 wt% of the self-assembling peptide mother solution was mixed with the above 2 wt% of laminin, fibronectin, fibrinogen, vitronectin, γ-globulin, transferrin, hemoglobin, and human collagen solutions in a 1:1 volume ratio to obtain a mixed system with a final concentration of 1 wt% of the self-assembling peptide.

[0267] To simultaneously measure the gel formation rate and strength of the mixed system, 200 μL of each sample was immediately added to the 20 mm diameter faceplate of a MARS 60 rheometer upon preparation. The final modulus was recorded after a 30-minute time scan. The distance between the rotor and the faceplate was 500 μm, the shear strain was 1%, and the frequency was 1 Hz. The entire test was completed at 37°C. The results of the mixed system formed by the self-assembling peptide represented by SEQ ID NO. 14 (FIFIFGTVIGPGGEGOGGV) with laminin, fibronectin, fibrinogen, vitronectin, transferrin, gamma globulin, hemoglobin, and human collagen are shown in Figure 7.

[0268] The results show that in each sample with a self-assembling peptide concentration of 1wt%, when the self-assembling peptide exists alone, although its storage modulus is higher than the loss modulus, there is no obvious difference, and both are lower than 2Pa (not shown in the figure), and the macroscopic appearance is liquid rather than solid; but when protein is present in the sample, the storage modulus (G') of the mixed system self-assembling peptide material of the present invention rises rapidly within 5 minutes, which is significantly higher than the loss modulus (G"), and forms a high-strength hydrogel with an elastic modulus greater than 100Pa within 30 minutes (Figure 7 shows G' and G" at 30 minutes). This shows that the self-assembling peptide molecules of the present invention are rapidly assembled to form a hydrogel with a certain mechanical strength under the initiation of positively charged natural substances (especially proteins with an isoelectric point PI of less than 7.0). However, due to its high isoelectric point, hemoglobin can only form a weaker gel, and the mixed solution of collagen cannot form a gel at all.

[0269] Experiment 2: Determination of mechanical strength of the hydrogel of the present invention

[0270] Experimental method: Same as Experiment 3, taking the mixed system formed by the self-assembling peptide represented by SEQ ID NO. 18 (IVIVIGSIIGOGGDGPGGV) and laminin, fibronectin, fibrinogen, and human serum albumin as an example, the results are shown in Figure 8.

[0271] The storage modulus (G') and loss modulus (G") measured after the polypeptide solution was mixed with different initiating proteins for 30 minutes are shown in Figure 8. The mechanical properties of the hydrogels formed by different proteins showed obvious differences. In all groups with the addition of the initiating protein of the present invention, hydrogels with a storage modulus greater than 300 Pa were observed to be formed in the mixed system, which is at least twice the storage modulus of the hydrogel formed by human serum albumin. This shows that the responsive hydrogels formed by the self-assembling peptides of the present invention and laminin, fibronectin, and fibrinogen have stronger mechanical strength, that is, stronger supporting function, than the responsive hydrogels formed by the self-assembling peptides and human serum albumin.

[0272] Experiment 3: Determination of the reassembly characteristics of the hydrogel after deformation

[0273] Experimental method: A shear thinning test was performed to verify the reassembly performance of the hydrogel of the present invention after deformation.

[0274] Preparation of hydrogel samples: 1 wt% self-assembling peptide solution was mixed with 1 wt% laminin, fibronectin, fibrinogen, vitronectin, γ-globulin, transferrin, hemoglobin, and human collagen solutions at a volume ratio of 1:1 to obtain a mixed system with a final concentration of 1 wt% self-assembling peptide.

[0275] During the test, 200 μL of the mixed system was added to the 20 mm diameter faceplate of a MARS 60 rheometer and a time sweep was performed. Shear thinning was then performed for 20 seconds with a rotor-faceplate spacing of 500 μm, a shear strain of 100%, and a frequency of 1 Hz. Shear thinning was then stopped, the shear strain immediately adjusted to 1%, and a time sweep test was immediately performed to monitor the recovery of the hydrogel's mechanical strength. Each 20-second shear thinning test constituted a single set of experiments, which were repeated twice to examine the reassembly ability of the hydrogels of the present invention after multiple external force-induced deformation. Using a mixed system formed by the self-assembling peptide represented by SEQ ID NO. 13 (IIIIIGOGIIGPGGEGPGGE) and tissue fluid (containing a large amount of proteins such as laminin, fibronectin, and vitronectin) as an example, the results are shown in Figure 9.

[0276] The results in Figure 9 show that the hydrogel self-assembled in response to tissue fluid exhibits the characteristic of being able to quickly re-self-assemble after being damaged and deformed by external force, and this characteristic does not disappear as the number of damages increases (Figure 9). When subjected to 100% shear strain, some non-covalent bonds inside the hydrogel, such as hydrogen bonds, break, causing its mechanical strength to decrease and become liquid; when the external force disappears, the partially destroyed nanofiber clusters will quickly approach and reassemble, quickly restoring their original mechanical strength. Figure 9 shows that after experiencing multiple shear thinning, the hydrogel of the present invention can still re-self-assemble, and the self-assembly time and the strength of the reorganized hydrogel are extended with only a small loss as the number of shear thinning increases.

[0277] Because laminin, fibronectin, vitronectin, and other proteins are abundant in tissue fluid, the self-assembling peptides of the present invention can rapidly initiate the formation of hydrogels. The resulting hydrogels exhibit shear-thinning and self-repairing properties, making them injectable. When used in cell culture, the hydrogels of the present invention enable multiple pipetting transfers, facilitating cell packaging and fluid replacement. Furthermore, when used in the biomedical field, they enable multiple injections.

[0278] 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.

[0279] Example 7: Application of the peptide hydrogel material of the present invention in the field of biomedicine

[0280] Experiment 1: The supporting effect of the hydrogel of the present invention on cells

[0281] Experimental method: Adherent HepG2 liver cancer cells were cultured in a cell culture flask in a mild and humid environment at 37°C and 5% CO2 using high-glucose DMEM medium containing 10% serum. After culture, the cells were collected and resuspended in PBS buffer solution to obtain a concentration of 5×10 5 cells / mL of cell suspension. The prepared cell suspension and Calcein-AM / PI staining solution were mixed evenly in a volume ratio of 2: 1 and incubated in the dark at 37°C for 15 minutes. A hydrogel containing fibrinogen, transferrin, and γ-globulin prepared in advance was added to a glass-bottomed culture dish (see Example 3 for the preparation method). The cell suspension after incubation in the dark was then added and mixed to make the total volume in the glass-bottomed culture dish 200 μL and the final concentration of the self-assembling peptide 0.1 wt%. When preparing the control group sample, an equal volume of PBS buffer was used to replace the hydrogel material of the present invention in the experimental group. Subsequently, each experimental sample was observed using an LSM 980 with Airyscan2 fast super-resolution laser confocal microscope. Taking the mixed system formed by the self-assembling peptide shown in SEQ ID NO.13 (IIIIIGOGIIGPGGEGPGGE) and fibrinogen, transferrin, and γ-globulin as an example, the results are shown in Figure 10.

[0282] The results in Figure 10 show that in the control group without the hydrogel of the present invention, the cells naturally sank to the bottom of the glass-bottom culture dish, were not supported, and were distributed in a 2D plane; when the self-assembling peptides were introduced into the system to form a hydrogel that self-assembled in response to fibrinogen, transferrin, and γ-globulin, the cells in the system were supported by the three-dimensional network scaffold formed by the self-assembling peptides, showing a three-dimensional distribution in a 3D environment.

[0283] Experimental Conclusion: This experimental phenomenon demonstrates that the hydrogel material of the present invention, namely, the three-dimensional network scaffold formed by the self-assembling peptides in response to proteins, can effectively support cells, facilitate the growth and cultivation of cells in three dimensions in vitro, and prevent them from sinking and adhering to the wall, thereby allowing them to grow in a two-dimensional environment. Furthermore, the entire initiation process is mild, requires no additional substances, and is effective, convenient, and harmless to cells. This demonstrates the enormous potential of the polypeptide material of the present invention in the fields of cell culture and biomedicine.

[0284] The results of experiments on self-assembling peptides shown by other sequence numbers are similar to those of this example and are not described in detail here.

[0285] Experiment 2: Application of 3D Cell Storage

[0286] Experimental method: This experiment takes the storage of mouse mesenchymal stem cells at 4°C as an example. The isolated mouse mesenchymal stem cells were pipetted evenly and 10 ml of equal volume was dispensed into cryopreservation tubes and pipetted evenly to make the cell concentration between 1×10 6 In the 3D group, mouse serum and 1% double-antibody storage solution in 50% MAP + 50% SFEM were added to the peptide SEQ ID NO: 20 (FLIVIGOGIIGOGGEGPGGE) to a final concentration of 0.05%. In the 2D group, mouse serum and 1% double-antibody storage solution in 50% MAP + 50% SFEM were added, and then the cells were stored in a 4°C refrigerator.

[0287] Cell viability test:

[0288] (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;

[0289] (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.

[0290] Experimental results: In order to understand the changes in the survival rate of mesenchymal cells during the storage period, we used the Calcein-AM / PI live / dead double staining kit to double-stain them and used a flow cytometer to detect them. As shown in Figure 11, starting from the third day of storage, the survival rate of mouse mesenchymal stem cells in the 3D storage group was significantly higher than that in the 2D storage group, with a significant statistical difference (79% vs. 67%). And on the fifth day of storage, it still had a high cell survival rate (71% vs. 43%), and the survival rate increased by nearly 30%. This shows that the storage effect of the mesenchymal stem cell storage system used in the present invention is better than traditional 2D storage, which greatly improves the cell survival rate.

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

[0292] Experiment 3: Application of 3D Culture of Liver Cancer Cells

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

[0294] Prepare the concentration of 1×10 6 The concentration of 1×10 cells / mL cell suspension was prepared by 5 Taking SEQ ID NO:3 (IIIIIGSIIGOGGEGPGGV) as an example, the concentration obtained is 1×10 5 A cell suspension of 10 cells / mL was inoculated into a 96-well plate containing the self-assembling peptide of the present invention (the final concentration of the self-assembling peptide in each well was 0.3 wt.%, 0.5 wt.%, and 0.7 wt.%). The culture medium in the upper portion of the plate was removed and replaced with fresh medium every 3 days during the culture period.

[0295] After 3, 7, 11, and 15 days of culture, the diameters of 50 cell spheres in the culture system were observed and recorded under an inverted microscope. The cell sphere diameter data collected on these three days were averaged and plotted into a graph (Figure 12). The HepG2 cells cultured in the self-assembling peptides of the present invention were tested to see whether they could achieve long-term in vitro proliferation, and characteristic cell spheres were photographed (Figure 12). As shown in Figure 12, in a culture system containing 0.3wt.%, 0.5wt.%, and 0.7wt.% of the triggered self-assembling peptides of the present invention, HepG2 cells were supported and subsequently grew and proliferated in the form of cell spheres. As the culture time prolonged, the cell spheres became increasingly compact and their diameters increased at a similar rate, indicating that HepG2 cells were able to proliferate stably at a uniform rate over a 15-day culture period. That is, the culture system of the present invention, which is triggered by a mixed system containing protein-containing substances and contains self-assembling peptides, can be used for long-term in vitro three-dimensional cell culture. The relatively uniform cell proliferation rate also indicates that it is in good condition during the culture period and does not cause damage to the cells.

[0296] 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.

[0297] Experiment 4: Application of 3D Culture of Porcine Muscle Satellites

[0298] The culture of porcine muscle satellite cells was similar to the 3D culture of liver cancer cells in Experiment 3. The concentration was prepared as in Experiment 2, 1×10 6 The concentration of 1×10 cells / mL cell suspension was prepared by 5 Taking SEQ ID NO: 21 (IVIVIGOGIIGOGGDGOGGV) as an example, the concentration obtained is 1×10 5 Cells / mL of cell suspension were inoculated into 96-well plates to which the self-assembling peptides of the present invention had been added (the final concentration of the self-assembling peptides in each well was 0.3 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.

[0299] The diameters of 50 cell spheres in the culture system were observed and recorded under an inverted microscope after 1, 2, 3, and 4 days of culture. The averaged sphere diameter data collected over these three days were calculated and plotted (Figure 13). This was used to test whether porcine muscle satellite cells cultured in the self-assembling peptides of the present invention could achieve long-term in vitro proliferation, and characteristic spheres were photographed (Figure 13). As shown in Figure 13, in a mixed culture system containing 0.3 wt.% of the polypeptides of the present invention and a protein-containing substance, porcine muscle satellite cells were supported and subsequently grew and proliferated in the form of cell spheres. As the culture time increased, the cell spheres became increasingly compact, and their diameters increased at a consistent rate. This indicates that porcine muscle satellite cells were able to proliferate stably and uniformly over the four-day culture period. Specifically, the self-assembling peptides of the present invention, after being triggered by the protein-containing mixture, formed a 3D support structure, which can be used for long-term in vitro three-dimensional cell culture. The relatively uniform cell proliferation rate also indicates that the cells were in good condition during the culture period and were not damaged.

[0300] Experiments 1-4 of this example demonstrate that the culture system of the protein-based self-assembling peptides of the present invention has excellent cell-supporting capacity. Experiment 2 also demonstrates that the protein-based self-assembling peptides of the present invention have good biocompatibility and can be used for cell storage. Experiments 3 and 4 respectively demonstrate that the addition of the self-assembling peptides of the present invention to cell culture media containing natural protein-based substances can highly replicate the in vivo cell growth environment, enabling long-term three-dimensional cell culture in vitro.

[0301] Example 8: Application of the hydrogel of the present invention as a dispersed filler for medical aesthetics

[0302] Experimental Method: The synthesized self-assembling peptide represented by SEQ ID NO. 25 (IIIIIGOGIIGOGGEGPGGV) was dissolved in deionized water and vortexed until fully dissolved to obtain a 1.5% self-assembling peptide solution. The pH of the solution was adjusted to 7.4 with 0.1M NaOH solution. Figure 14A shows, from left to right, the peptide solution, the precipitated L-polylactic acid microsphere solution, and a mixture of the two. The peptide solution was autoclaved and then mixed with L-polylactic acid microspheres (particle size between 25-60 μm). Finally, the solution was aliquoted to obtain a 5% microsphere-containing self-assembling peptide mixed injection for injection. The solution became opaque, and the microspheres were evenly and stably distributed in the self-assembling peptide solution (Figure 14B). The resulting microsphere-containing self-assembling peptide mixed injection was stored at room temperature for one month. The obtained injections were uniformly suspended, with no obvious solid-liquid separation. After adding 1 / 5 tissue fluid, hydrogel formation was observed (Figure 14C).

[0303] Similarly, using the same method and steps, SEQ ID NO. 27 (FLIVIGSIIGOGAEGPGGV) at a concentration of 1.5% can also achieve support for 5% polycaprolactone (PCL) microspheres. As shown in Figure 15A, from left to right, there are a transparent SEQ ID NO. 27 polypeptide solution, a precipitated polycaprolactone (PCL) microsphere solution, and a mixture of the two. The high-temperature sterilized polypeptide solution was mixed with polycaprolactone (PCL) microspheres (particle size between 25-50um) to make the content of polycaprolactone (PCL) microspheres 5%. It can be seen that the microspheres are uniformly and stably distributed in the self-assembling peptide solution (Figure 15B). The obtained self-assembling peptide mixed injection containing microspheres was placed at room temperature for one month. It can be seen that the obtained injections are all in a uniform suspension state, with no obvious solid-liquid separation phenomenon. After adding 1 / 5 tissue fluid, a gel formation phenomenon can be seen (Figure 15C).

[0304] In addition, except for SEQ ID NOs. 25 and 27, the self-assembling peptides represented by SEQ ID NOs. 1-7 and 9-32 of the present invention can have the same function on a variety of other medical aesthetic regeneration microspheres (Table 2).

[0305] Table 2 Common medical aesthetic microspheres:

[0306] Experimental conclusion: This experimental phenomenon proves that the hydrogel material of the present invention, that is, the three-dimensional network scaffold self-assembled by self-assembling peptides in response to proteins, can effectively support medical aesthetic microspheres. It can play a good role in dispersing and supporting the microspheres when it is not activated. After being activated by proteins in the tissue fluid, it can quickly form a hydrogel, thereby playing a local shaping function in the tissue; the entire experimental process does not require the addition of other ingredients, and the operation is convenient.

[0307] 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.

[0308] 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 composition comprising a self-assembling peptide and microspheres, wherein the self-assembling peptide comprises a hydrophobic domain and a hydrophilic domain, wherein the hydrophilic domain comprises at least two consecutive β-turn regions.

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

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

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

7. The composition 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 composition according to claim 3, wherein one or more of X1, X3 and X4 is glycine, and / or one or both of X3 and X4 is alanine (A).

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

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

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

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

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

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

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

16. The composition according to claim 1, wherein the microspheres are selected from the group consisting of poly (L-lactic acid) microspheres, polycaprolactone microspheres, poly (lactic-co-glycolic acid) microspheres, hydroxyapatite microspheres, poly (methyl methacrylate) microspheres and polyvinyl alcohol microspheres.

17. The composition according to claim 1, wherein the particle size of the microspheres is 3-150 μm; Preferably, the particle size of polylactic acid microspheres is 10-60 μm, the particle size of polycaprolactone microspheres is 10-60 μm, the particle size of polylactic acid-glycolic acid microspheres is 20-60 μm, the particle size of hydroxyapatite microspheres is 25-45 μm, and the particle size of polymethyl methacrylate microspheres is 20-60 μm.

18. The composition according to claim 1, further comprising a positive charge source as an initiator, wherein the positive charge source is a biomacromolecule, a drug, a functional molecule, a metal ion, an amino acid, or a mixture comprising one or more endogenous or exogenous substances thereof. The biomacromolecules are selected from organic acids, proteins, polysaccharides and their derivatives, Preferably, the organic acid is selected from lactic acid, tannic acid and citric acid; Preferably, the protein is a protein that can provide hydrogen ions under neutral physiological conditions. Preferably, the protein has an isoelectric point PI value lower than 7.0, preferably 3.4-6.

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

19. The composition according to claim 1, wherein the self-assembling peptide and the microspheres are placed in the same container in the form of powder; or, the self-assembling peptide and the microspheres 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 microspheres are placed in another container in the form of a powder or solution; or, the self-assembling peptide is placed in one container in the form of a powder, and the microspheres are placed in another container in the form of a solution; or the peptide and the microspheres are placed in one container in the form of a solution.

20. A method of forming a microsphere filler from the composition of any one of claims 1 to 19, the method comprising: The invention also comprises the steps of mixing the microspheres with the self-assembling peptides and inducing the self-assembling peptides to form a three-dimensional network scaffold material under the conditions of a positive charge source.

21. The method according to claim 20, wherein the initiating self-assembling peptides to form a three-dimensional network scaffold material under the conditions of a positive charge source comprises: Initiation is performed at a pH less than 6; or, At pH 6-10, the reaction is initiated by a positive charge source. Alternatively, a solution of self-assembling peptides and microspheres is injected into animal or human tissue, where it is triggered by the positive charge source.

22. The method according to claim 21, wherein the positive charge source is a biomacromolecule, a drug, a functional molecule, a metal ion, an amino acid, or a mixture comprising one or more endogenous or exogenous substances thereof. The biomacromolecules are selected from organic acids, proteins, polysaccharides and their derivatives, Preferably, the organic acid is selected from lactic acid, tannic acid and citric acid; Preferably, the protein is a protein that can provide hydrogen ions under neutral physiological conditions. Preferably, the protein has an isoelectric point PI value lower than 7.0, preferably 3.4-6.

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

23. A microsphere filler comprising the composition of any one of claims 1 to 19, or prepared by the method of any one of claims 20 to 22.

24. The microsphere filler according to claim 23, wherein the microsphere filler is a polypeptide solution, polypeptide powder, a three-dimensional network scaffold material in the form of a hydrogel, or a dry hydrogel three-dimensional network scaffold material.

25. Use of the composition of any one of claims 1-19, the method of any one of claims 20-22, or the microsphere filler of claim 23 or 24 in one or more selected from the group consisting of: regenerative medicine and tissue regeneration; drug delivery; wound healing; implantable materials; gene therapy; stem cell therapy; and medical aesthetics.

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