Multifunctional self-assembly oligopeptide ZT48, preparation method and application thereof, and oligopeptide ZT48 self-assembly body

Abstract

The invention relates to the technical field of biological medicine, in particular to a multifunctional self-assembled oligopeptide ZT48, a preparation method and application thereof and an oligopeptide ZT48 self-assembly, according to the multifunctional self-assembled oligopeptide ZT48, tyrosine is fixed to serve as a tail, phenylalanine and tryptophan are connected to serve as a head, a benzene ring structure is provided for the whole molecular structure, and the oligopeptide ZT48 self-assembly is obtained. Self-assembly can be carried out through a pi-pi stacking effect; five amino acids in the middle of the molecule are respectively represented by residues R1, R2, R3, R4 and R5, R1-R5 are alternately connected through hydrophobic and hydrophilic amino acids, hydrophilic residues are utilized to provide hydrophilicity for the oligopeptide molecule, hydrogen bonds can be formed between the oligopeptide molecule and water molecules, and meanwhile, free amino groups, imino groups and hydroxyl groups on the amino acid residues in the polypeptide molecule can be used for preparing the oligopeptide molecule. The hydrophobic residues form hydrogen bonds with oxygen atoms on carbonyl to regulate and control polypeptide molecules to generate self-assembly, the hydrophobic residues provide hydrophobicity for short peptide molecules, and through the hydrophobic effect, molecules are orderly arranged, and the self-assembly behavior of the polypeptide molecules is regulated and controlled.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and in particular to a multifunctional self-assembling short peptide ZT48, its preparation method and application, and a short peptide ZT48 self-assembly Background Art

[0002] Polypeptides are compounds formed by connecting peptide chains through dehydration condensation of multiple amino acids. They are widely present in organisms and are the basis of life activities and the development of biological materials. In addition, polypeptides are also important substances for regulating the functions of the body. In recent decades, polypeptide molecules have been used clinically in applications such as targeted cancer therapy, synthesis of key drugs for treating diabetes and osteoporosis, and in vitro injection by mimicking hormones. However, with the further development of polypeptide drugs, bottlenecks in the development of polypeptide drugs have emerged. Disadvantages such as poor stability, high cytotoxicity, and short half-life of monomeric polypeptide drugs limit their clinical applications. In recent years, the design and synthesis of short peptides with self-assembling properties have been emerging technical means and research directions in the field of polypeptides. The modification of monomeric molecules into macromolecular aggregates with specific nanostructures has been widely studied to optimize these problems and expand the future development prospects of polypeptide drugs

[0003] Short peptides have a small molecular weight, and their specific molecular structures endow them with excellent designability. They have simple structures, are easy to chemically modify, and have good biocompatibility and biodegradability. Their assemblies show potential application prospects in drug delivery, cell culture, and tissue engineering and regenerative medicine. However, existing short peptides have relatively single functionality, narrow application fields, and there is a potential risk of triggering immune responses in polypeptide molecules with more than 9 amino acids

[0004] Therefore, the existing technology still needs to be improved and developed Summary of the Invention

[0005] In view of the above deficiencies of the existing technology, the purpose of the present invention is to provide a multifunctional self-assembling short peptide ZT48, its preparation method and application, and a short peptide ZT48 self-assembly, aiming to solve the problems of relatively single functionality, narrow application fields of existing short peptides, and the potential risk of triggering immune responses in polypeptide molecules with more than 9 amino acids

[0006] The technical solution of the present invention is as follows

[0007] A multifunctional self-assembling short peptide ZT48, whose chemical structural general formula is shown as follows

[0008]

[0009] Among them, R in the formula 1 、R 3 、R 5 are hydrophobic residues, R2 , R 4 is a hydrophilic residue.

[0010] For the multifunctional self-assembling short peptide ZT48, wherein the R 1 , the R 3 and the R 5 are independently selected from one of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, tryptophan.

[0011] For the multifunctional self-assembling short peptide ZT48, wherein the R 2 and the R 4 are independently selected from one of arginine, lysine, histidine, aspartic acid, glutamic acid; the R 2 and the R 4 carry opposite charges.

[0012] For the multifunctional self-assembling short peptide ZT48, wherein the chemical structural formula of the multifunctional self-assembling short peptide ZT48 is:

[0013]

[0014] A preparation method of a multifunctional self-assembling short peptide ZT48, comprising the steps:

[0015] Use an eluent to remove Fmoc from Fmoc-Phe-Wang Resin, and after solid-liquid separation, obtain a first solid-phase substance;

[0016] Mix the first solid-phase substance with Fmoc-Trp(Boc)-OH for a reaction, then use an eluent to remove Fmoc, and after solid-liquid separation, obtain a second solid-phase substance;

[0017] Add an amino acid containing R 1 to the second solid-phase substance for a reaction, and after the reaction, use an eluent to remove Fmoc, and then use amino acids containing R 2 -R 5 in sequence to complete the reaction and remove Fmoc, and after solid-liquid separation, obtain a third solid-phase substance;

[0018] Mix the third solid-phase substance with Fmoc-Tyr(tBu)-OH for a reaction, then use an eluent to remove Fmoc, and after solid-liquid separation, obtain a fourth solid-phase substance;

[0019] Use acetic anhydride to perform N-terminal acetylation treatment on the fourth solid-phase substance to obtain a resin;

[0020] The resin is subjected to a cleavage reaction with a cleavage solution to obtain a reaction residue, and the reaction residue is sedimented with ice isopropyl ether to obtain the multifunctional self-assembling short peptide ZT48.

[0021] The preparation method of the multifunctional self-assembling short peptide ZT48, wherein the eluent is composed of diethylamine dissolved in N,N-dimethylformamide; the concentration of the eluent is 5wt%-30wt%.

[0022] The preparation method of the multifunctional self-assembling short peptide ZT48, wherein after the solid-liquid separation step, it includes a washing treatment; the solvents used for the washing treatment include one or more of methanol, ethanol, DMF, and DCM.

[0023] The preparation method of the multifunctional self-assembling short peptide ZT48, wherein the cleavage solution is one or more of TFA, H 2 O, Tis, and DODT; the time of the cleavage reaction is 1h-3h.

[0024] An application of the multifunctional self-assembling short peptide ZT48 in the preparation of cosmetics, biomedical nanomaterials, tissue engineering materials, and drug delivery carriers.

[0025] A short peptide ZT48 self-assembly, which is obtained by the intramolecular or intermolecular self-assembly of the multifunctional self-assembling short peptide ZT48.

[0026] Beneficial effects: The present invention provides a multifunctional self-assembling short peptide ZT48 and its preparation method and application, and a short peptide ZT48 self-assembly. The multifunctional self-assembling short peptide ZT48 fixes tyrosine as the tail, and phenylalanine and tryptophan are connected as the head, providing a benzene ring structure for the entire molecular structure, enabling it to self-assemble through π-π stacking interactions; the 5 amino acids in the middle of the molecule have their residues R 1 、R 2 、R 3 、R 4 and R 5 represented by, R 1 ~R 5By alternately connecting hydrophobic and hydrophilic amino acids, hydrophilic residues are used to provide hydrophilicity for the short peptide molecules, which can form hydrogen bonds with water molecules. At the same time, in the polypeptide molecules, hydrogen bonds can be formed between the free amino groups, imino groups and hydroxyl groups on the amino acid residues and the oxygen atoms on the carbonyl groups to regulate the self-assembly of the polypeptide molecules. The hydrophobic residues provide hydrophobicity for the short peptide molecules, and through hydrophobic interactions, the molecules are arranged orderly to regulate the self-assembly behavior of the polypeptide molecules. In addition, among the five amino acids in the middle of the molecule, two hydrophilic residues carry opposite charges, providing electrostatic interactions for the short peptide molecules, enabling them to self-assemble through electrostatic interactions. In addition, the nano-network self-assembly formed by the multifunctional self-assembling short peptide ZT48 through the hydrogen bond network can provide a scaffold for cells, promote cell adhesion, simulate the extracellular matrix conditions, thereby promoting cell proliferation and migration, and promoting the repair of the cell barrier. Description of the Drawings

[0027] Figure 1 Schematic process flow diagram of the preparation method of a multifunctional self-assembling short peptide ZT48 of the present invention;

[0028] Figure 2 Mass spectrometry spectrum of ZT48 prepared in Example 1 of the present invention;

[0029] Figure 3 High performance liquid chromatography (HPLC) purity detection spectrum of ZT48 prepared in Example 1 of the present invention;

[0030] Figure 4 Morphology diagram of the self-assembly of ZT48 prepared in Example 1 of the present invention;

[0031] Figure 5 Circular dichroism spectrum of the self-assembly of ZT48 prepared in Example 1 of the present invention;

[0032] Figure 6 Result diagram of promoting cell proliferation of ZT48 prepared in Example 1 of the present invention;

[0033] Figure 7 Result diagram of promoting cell migration of ZT48 prepared in Example 1 of the present invention;

[0034] Figure 8 Result diagram of promoting cell adhesion of ZT48 prepared in Example 1 of the present invention;

[0035] Figure 9 Result diagram of the relative expression of genes promoting the repair of the keratinocyte barrier of ZT48 prepared in Example 1 of the present invention. Detailed Description of the Invention

[0036] The present invention provides a multifunctional self-assembling short peptide ZT48, its preparation method and application, and a self-assembly of short peptide ZT48. To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention will be further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0037] Those skilled in the art of the present technology can understand that, unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as the general understanding of those of ordinary skill in the field to which the present invention belongs. It should also be understood that terms such as those defined in a general dictionary should be understood to have a meaning consistent with the meaning in the context of the prior art, and will not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0038] Self-assembling short peptides can rely on weak intermolecular interactions to spontaneously or triggeredly bind and connect in a system, thereby forming nanoscale structural aggregates visible under a microscope. These nanostructures include spherical micelles, vesicles, linear, ribbon-like, lamellar, columnar, tubular, spherical and reticular structures, etc. In addition, the self-assembled nanostructures thereof can also form hydrogels. The high biocompatibility and good biological activity exhibited by these aggregates have been used for the construction of multi-purpose modules and nanodevices. It can be seen that self-assembling short peptides have shown more diverse functionality than monomeric polypeptide molecules, and the development of self-assembling short peptides has become an inevitable trend.

[0039] Based on this, the present invention provides a multifunctional self-assembling short peptide ZT48, and its chemical structural general formula is as follows:

[0040]

[0041] Among them, R in the formula 1 , R 3 , R 5 are hydrophobic residues, and R 2 , R 4 are hydrophilic residues.

[0042] In this embodiment, the multifunctional self-assembling short peptide ZT48 fixes tyrosine as the tail, and phenylalanine and tryptophan are connected as the head, providing a benzene ring structure for the entire molecular structure, enabling it to self-assemble through π-π stacking; the 5 amino acids in the middle of the molecule are represented by their residues R 1 , R 2 , R 3 , R 4 and R 5 , R 1 ~R 5 are alternately connected by hydrophobic and hydrophilic amino acids (i.e., R 1: Hydrophobic, R 2 : Hydrophilic, R 3 : Hydrophobic, R 4 : Hydrophilic, R 5 : (Hydrophobic). By using hydrophilic residues to provide hydrophilicity for the short peptide molecule, hydrogen bonds can be formed with water molecules. At the same time, in the polypeptide molecule, hydrogen bonds (i.e., N-H…O=C, O-H…O=C) can be formed between the free amino group, imino group and hydroxyl group on the amino acid residue and the oxygen atom on the carbonyl group, regulating the self-assembly of the polypeptide molecule. The hydrophobic residues provide hydrophobicity for the short peptide molecule, and through hydrophobic interaction, the molecules are arranged orderly, regulating the self-assembly behavior of the polypeptide molecule. In addition, among the 5 amino acids in the middle of the molecule, two hydrophilic residues carry opposite charges, providing electrostatic interaction for the short peptide molecule, enabling it to self-assemble through electrostatic interaction. In addition, the nano-network self-assembly formed by the multifunctional self-assembling short peptide ZT48 through the hydrogen bond network can provide a scaffold for cells, promote cell adhesion, simulate the extracellular matrix conditions, thereby promoting cell proliferation and migration, and promoting the repair of the cell barrier.

[0043] Specifically, the present invention provides a short peptide with self-assembly performance, which is an octapeptide molecule containing 8 amino acids composed of hydrophilic amino acids, hydrophobic amino acids and charged amino acids. The amino acid molecules are connected by amide bonds. The hydrophilicity and hydrophobicity as well as the charge of the molecule can provide non-covalent bond interactions such as hydrogen bond interaction, electrostatic interaction, hydrophobic interaction and π-π stacking to regulate the self-assembly of the polypeptide molecule and form a nano-network structure. Among them, the charged unit can provide the charge property for the polypeptide molecule and enhance the stability of the self-assembled nanostructure. And ZT48 is composed of amino acids and has good biocompatibility. Its nano-network self-assembly formed through the hydrogen bond network can provide a scaffold for cells, promote cell adhesion, simulate the extracellular matrix conditions, thereby promoting cell proliferation and migration, and promoting the repair of the cell barrier.

[0044] In some embodiments, the R 1 、the R 3 and the R 5 The amino acids where they are located are independently selected from one of glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), tryptophan (Trp, W). The above amino acids are all hydrophobic amino acids, which can provide hydrophobicity for the short peptide molecule. Through hydrophobic interaction, the molecules are arranged orderly, regulating the self-assembly behavior of the polypeptide molecule.

[0045] In some embodiments, the R 2 and the R 4The amino acids therein are independently selected from one of arginine (Arg, R), lysine (Lys, K), histidine (His, H), aspartic acid (Asp, D), and glutamic acid (Glu, E); the R 2 and the R 4 carry opposite charges. The above amino acids are all hydrophilic amino acids, which can provide hydrophilicity for short peptide molecules and can form hydrogen bonds with water molecules. At the same time, in the polypeptide molecule, hydrogen bonds (i.e., N-H…O=C, O-H…O=C) can be formed between the free amino group, imino group, and hydroxyl group on the amino acid residue and the oxygen atom on the carbonyl group to regulate the self-assembly of the polypeptide molecule.

[0046] In some embodiments, the chemical structural formula of the multifunctional self-assembling short peptide ZT48 is:

[0047]

[0048] In addition, as Figure 1 shown, the present invention also provides a preparation method of the multifunctional self-assembling short peptide ZT48, including the steps:

[0049] Step S10: Remove Fmoc from Fmoc-Phe-Wang Resin using an eluent, and after solid-liquid separation, obtain a first solid-phase substance;

[0050] Step S20: Mix the first solid-phase substance with Fmoc-Trp(Boc)-OH for a reaction, then remove Fmoc using an eluent, and after solid-liquid separation, obtain a second solid-phase substance;

[0051] Step S30: Add an amino acid containing R 1 to the second solid-phase substance for a reaction, and after the reaction, remove Fmoc using an eluent, and then use amino acids containing R 2 -R 5 in sequence to complete the reaction and remove Fmoc, and after solid-liquid separation, obtain a third solid-phase substance;

[0052] Step S40: Mix the third solid-phase substance with Fmoc-Tyr(tBu)-OH for a reaction, then remove Fmoc using an eluent, and after solid-liquid separation, obtain a fourth solid-phase substance;

[0053] Step S50: Perform N-terminal acetylation treatment on the fourth solid-phase substance using acetic anhydride to obtain a resin;

[0054] Step S60: Perform a cleavage reaction on the resin with a cleavage solution to obtain a reaction residue, and use ice isopropyl ether to sediment the reaction residue to obtain the multifunctional self-assembling short peptide ZT48.

[0055] In this embodiment, the multifunctional self-assembling short peptide ZT48 has tyrosine fixed as the tail and phenylalanine and tryptophan linked as the head, providing a benzene ring structure for the entire molecular structure, enabling it to self-assemble through π-π stacking interactions; the 5 amino acids in the middle of the molecule have their residues R 1 、R 2 、R 3 、R 4 and R 5 represented, R 1 ~R 5 are alternately connected by hydrophobic and hydrophilic amino acids. The hydrophilic residues provide hydrophilicity for the short peptide molecule, enabling the formation of hydrogen bonds with water molecules. At the same time, in the polypeptide molecule, hydrogen bonds can be formed between the free amino groups, imino groups, and hydroxyl groups on the amino acid residues and the oxygen atoms on the carbonyl groups to regulate the self-assembly of the polypeptide molecule. The hydrophobic residues provide hydrophobicity for the short peptide molecule, and through hydrophobic interactions, the molecules are arranged orderly to regulate the self-assembly behavior of the polypeptide molecule; in addition, among the 5 amino acids in the middle of the molecule, two hydrophilic residues carry opposite charges, providing electrostatic interactions for the short peptide molecule, enabling it to self-assemble through electrostatic interactions. In addition, the nano-network-like self-assembly formed by the multifunctional self-assembling short peptide ZT48 through a hydrogen bond network can provide a scaffold for cells, promote cell adhesion, simulate the extracellular matrix conditions, thereby promoting cell proliferation and migration, and promoting the repair of the cell barrier. Moreover, the short peptide prepared by this preparation method has self-assembly performance, and a significant nano-network structure is observed under the TEM electron microscope.

[0056] In some embodiments, the eluent is composed of diethylamine dissolved in N,N-dimethylformamide; the concentration of the eluent is 5wt%-30wt%. The eluent can be used to remove the Fmoc in the molecule, facilitating the connection of the next amino acid.

[0057] In a preferred embodiment, the concentration of the eluent is 10wt%-20wt%.

[0058] Specifically, the preparation method of the multifunctional self-assembling short peptide ZT48 specifically includes the steps:

[0059] 1) Preparation of Phe: Take Fmoc-Phe-Wang Resin and add it to the reactor. Remove Fmoc with a diethylamine / DMF solution, perform solid-liquid separation to obtain the solid phase material, wash the obtained solid phase material, and the ninhydrin test is positive;

[0060] 2) Coupling of Trp: Add Fmoc-Trp(Boc)-OH to the reactor containing the solid phase material in step (1) for reaction, then remove Fmoc with a diethylamine / DMF solution, perform solid-liquid separation to obtain the solid phase material, wash the obtained solid phase material, and the ninhydrin test is positive;

[0061] 3) Coupling of amino acids containing R 1 ~R 5 : Replace the amino acid raw materials, repeat step (2), and sequentially couple the amino acids containing R 1 ~R 5 : Sequentially add the preselected amino acids (each containing R 1 ~R 5 ) to the reactor containing the solid phase substance and react; then perform Fmoc deprotection with diethylamine / DMF solution, separate the solid and liquid phases, take the solid phase substance, wash the obtained solid phase substance, and the ninhydrin test is positive;

[0062] 4) Coupling of Tyr: Add Fmoc-Tyr(tBu)-OH to the reactor containing the solid phase substance in step (3) and react; then perform Fmoc deprotection with diethylamine / DMF solution, separate the solid and liquid phases, take the solid phase substance, wash the obtained solid phase substance, and the ninhydrin test is positive; Acetylate the N-terminus with acetic acid. Take the reaction product, wash and dry it to obtain the resin;

[0063] 5) Lyse the resin obtained in step (4) with the lysis solution to obtain the reaction residue, precipitate the obtained reaction residue with ice isopropyl ether, and separate and purify to obtain the self-assembled short peptide ZT48, whose amino acid sequence is Ac-Tyr-R5-R4-R3-R2-R1-Trp-Phe-OH.

[0064] In some embodiments, the method of solid-liquid separation includes one of filtration, centrifugation, and suction filtration; the above solid-liquid separation methods can maximize the separation of the solid phase substance. Preferably, the method of solid-liquid separation is suction filtration.

[0065] In some embodiments, after the step of solid-liquid separation, it includes a washing treatment; the solvents used in the washing treatment include one or more of methanol, ethanol, DMF, and DCM. The above solvents can remove impurities in the solid phase substance and improve the purity of the solid phase substance.

[0066] In some embodiments, the lysis solution is one or more of TFA, H 2 O, Tis, and DODT; the lysis reaction time is 1h - 3h.

[0067] In a preferred embodiment, the lysis solution is composed of a mixture of TFA, H 2 O, Tis, and DODT, and its volume percentage ratio is TFA:H 2 O:Tis:DODT = 92.5:2.5:2.5:2.5, and the lysis time is 1h.

[0068] In addition, the present invention also provides an application of a multifunctional self-assembling short peptide ZT48 in the preparation of cosmetics, biomedical nanomaterials, tissue engineering materials, and drug delivery carriers.

[0069] In this embodiment, the nano-network self-assembled body formed by the multifunctional self-assembling short peptide ZT48 through a hydrogen bond network can provide a scaffold for cells, promote cell adhesion, simulate the extracellular matrix conditions, thereby promoting cell proliferation and migration, and promoting the repair of the cell barrier. Therefore, it can be applied in fields such as cosmetic efficacy raw materials, biomedical nanomaterials, tissue engineering materials, and drug delivery carriers.

[0070] In some embodiments, the multifunctional self-assembling short peptide ZT48 can be used to promote cell migration, promote cell proliferation, promote cell adhesion, and promote the repair of the keratinocyte skin barrier; specifically, the effective concentration for promoting cell migration is 50-1000 ppm, the effective concentration for promoting cell proliferation is 25-1000 ppm, the effective concentration for promoting cell adhesion is 50-1000 ppm, and the effective concentration for promoting the repair of the keratinocyte skin barrier is 50-1000 ppm.

[0071] In addition, the present invention also provides a short peptide ZT48 self-assembled body, which is obtained by the self-assembly of the multifunctional self-assembling short peptide ZT48 through intramolecular or intermolecular self-assembly.

[0072] In this embodiment, the nano-network self-assembled body formed by the multifunctional self-assembling short peptide ZT48 through a hydrogen bond network can provide a scaffold for cells, promote cell adhesion, simulate the extracellular matrix conditions, thereby promoting cell proliferation and migration, and promoting the repair of the cell barrier.

[0073] The following further gives examples to illustrate the present invention in detail. Similarly, it should be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the protection scope of the present invention.

[0074] Example 1

[0075] In this example, a multifunctional self-assembling short peptide ZT48 is prepared as follows:

[0076] Materials used in this example: Fmoc-Phe-OH (fluorenylmethoxycarbonyl-phenylalanine), Fmoc-Trp(Boc)-OH (fluorenylmethoxycarbonyl-tryptophan-ε-tert-butoxycarbonyl), Fmoc-Leu-OH (fluorenylmethoxycarbonyl-leucine), Fmoc-Glu(OtBu)-OH (fluorenylmethoxycarbonyl-glutamic acid-1-tert-butyl ester), Fmoc-Ile-OH (fluorenylmethoxycarbonyl-isoleucine), Fmoc-Lys(Boc)-OH (fluorenylmethoxycarbonyl-lysine-ε-tert-butoxycarbonyl), Fmoc-Pro-OH (fluorenylmethoxycarbonyl-proline), Fmoc-Tyr(tBu)-OH (fluorenylmethoxycarbonyl-tyrosine-O-tert-butyl), Wang resin, HOBT (1-hydroxybenzotriazole), DIC (N,N'-diisopropylcarbodiimide), DMAP (4-dimethylaminopyridine), NMM (N-methylmorpholine), diethylamine, acetic anhydride; solvents: DMF (N,N-dimethylformamide), DCM (dichloromethane), TFA (trifluoroacetic acid), TIS (triisopropylsilane), DODT (2,2'-(ethylenedioxy)bis(ethanethiol)), ACN (acetonitrile), isopropyl ether.

[0077] The specific operation process of solid-phase synthesis of ZT48 protected by Fmoc (fluorenylmethoxycarbonyl) is as follows:

[0078] (1) Coupling of the first Phe to Wang resin: Weigh an appropriate amount of Wang resin and place it in a peptide synthesis column. Swell it with DCM (200 ml) for 30 - 40 min, drain the solvent. Weigh 31.48 g of Fmoc-Phe-OH and 10.98 g of HOBT, dissolve them in 100 ml of DMF, pre-activate in a -20°C refrigerator for 20 - 30 min, add 10.26 g of DIC and 1 g of DMAP, stir to dissolve, and add to the peptide synthesis column for reaction for 2 - 3 h. After the reaction, drain the solvent, wash twice with DMF and once with DCM in sequence. Add 150 ml of a mixed solution containing propionic anhydride (2.54 g), DMAP (0.24 g), and NMM (0.66 g), react for 1 h, drain the solvent, wash twice with DMF and once with DCM in sequence. Add 150 ml of 20% diethylamine / DMF solution, react for 30 - 40 min, drain the solvent, wash once with DMF and once with DCM, and the ninhydrin test shows positive.

[0079] (2) Coupling of the second Trp: Add the raw materials Fmoc-Trp(Boc)-OH (6.85 g), HOBT (1.76 g), DIC (1.64 g), and DMF (100 ml) to the reactor, react for 1 - 2 h. Take a small amount of resin for ninhydrin detection, and the result is negative, indicating that the reaction is complete. After stopping the reaction, drain the solvent, and wash the obtained resin twice with DMF and once with DCM. Add 150 ml of 20% diethylamine / DMF solution, react for 30 - 40 min, drain the solvent, and wash once with DMF and once with DCM. The ninhydrin test shows a positive result.

[0080] (4) Coupling of the third to the eighth amino acids: Repeat step (2) to couple Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH in sequence. After the coupling of the eighth amino acid is completed, add acetic anhydride to acetylate the -NH at the N-terminus. 2 Perform acetylation treatment.

[0081] (5) After the reaction is completed, drain the solvent, wash twice with 150 ml of DMF, once with 150 ml of DCM, once with 150 ml of methanol for shrinkage, once with 150 ml of DCM, and once with 150 ml of methanol for shrinkage. Dry overnight in a vacuum at 39 °C.

[0082] (6) Use the cleavage solution TFA:H 2 O:Tis:DODT = 92.5:2.5:2.5:2.5. Cleave the above resin with 150 ml for 1 - 3 h. After concentrating and removing most of the TFA with a rotary evaporator, add it to 10 times the volume of ice-cold isopropyl ether for precipitation, and filter to obtain the crude peptide ZT-48 as a solid.

[0083] Purification steps:

[0084] (1) First, analyze the purity of the obtained crude peptide above. After determining the retention time of the main peak, analyze the sample by linear gradient;

[0085] (2) Weigh 50 mg of the crude peptide, dissolve and dilute it to 3 ml with the mobile phase. Filter it with a 0.45 μm organic filter membrane, take the subsequent filtrate, and perform gradient separation with a high-performance liquid chromatograph. Collect the corresponding mobile phase at the retention time of the target absorption peak to obtain the ZT48 solution. Among them, the results of the HPLC gradient analysis method are shown in Table 1.

[0086] Chromatographic column: C18 chromatographic column (4.6×250 mm, 5 μm)

[0087] Mobile phase A: 0.1% trifluoroacetic acid / aqueous solution

[0088] Flow B: Acetonitrile

[0089] Flow rate: 1 ml / min

[0090] Column temperature: 30 °C

[0091] Detection wavelengths: 250 nm, 210 nm

[0092] Injection volume: 5 μL

[0093] Table 1 HPLC gradient analysis method

[0094] Experiment time Mobile phase A Mobile phase B 0 - 5 min 95％ 5％ Equilibration 5 - 30 min 5％ 95％ Gradient 30 min - 40 min 95％ 5％ Equilibration 40 min - 45 min 95％ 5％ Equilibration

[0095] (3) Place 500 mg of the sample in a 20 ml dissolution flask, add deionized water, dissolve it by ultrasonic treatment, filter it with a 0.45 μm organic phase filter head, and take the subsequent filtrate;

[0096] (4) Inject it into a high performance liquid chromatograph, prepare it with a linear gradient, collect the target peak, and determine whether the MS is correct;

[0097] (5) To meet the purity requirements of the liquid volume, track and feedback the purity of the collected liquid;

[0098] (6) At 40 °C, concentrate the collected qualified solution to 40 - 80 mL;

[0099] Salt conversion step:

[0100] Add ammonium acetate with a molar amount 2 - 3 times the number of free amino groups in ZT48 to the purified and concentrated sample, dissolve it thoroughly by ultrasonic treatment, inject it into a high performance liquid chromatograph, prepare it with a linear gradient, and collect the target peak. At 40 °C, concentrate the collected solution to 40 - 80 ml, and freeze-dry to obtain a white powdery solid ZT48.

[0101] Example 2

[0102] ZT48 mass spectrometry detection:

[0103] Perform mass spectrometry testing on the ZT48 prepared in Example 1. The test results are shown in Figure 2 , and the experiment proves the successful synthesis of ZT48.

[0104] Example 3

[0105] ZT48 purity detection:

[0106] Perform high performance liquid chromatography (HPLC) detection on the ZT48 prepared in Example 1. The test results are shown in Figure 3 , and determine its purity to be 95.4% according to the peak area in the figure.

[0107] Example 4

[0108] Observation of the self-assembled morphology of ZT48:

[0109] Weigh an appropriate amount of the self-assembled short peptide ZT48 prepared in Example 1 accurately, dissolve it in ultrapure water to make the concentration of ZT48 2 mg / ml, sonicate for 10 min to completely dissolve it, transfer the ZT48 solution to 37 °C and let it stand for 3 days. Take 10 μl of the above ZT48 solution, dilute it 5 times with ultrapure water, drop it on a carbon-supported copper grid (200 mesh), let it stand and settle for 30 min, then use a disposable filter paper to suck away the liquid drop, air-dry it overnight naturally, and observe and photograph it using a biological transmission electron microscope.

[0110] The self-assembled morphology of ZT48 is as Figure 4 shown. The self-assembled solution of ZT48 was observed under a transmission electron microscope and was found to exhibit an obvious nano-network structure.

[0111] Example 5

[0112] Detection of the self-assembled secondary structure of ZT48:

[0113] Weigh an appropriate amount of the self-assembled short peptide ZT48 prepared in Example 1 accurately, dissolve it in ultrapure water to make the concentration of ZT48 1 mg / ml, sonicate for 10 min to completely dissolve it, transfer the ZT48 solution to 37 °C and let it stand for 3 days. Take the above ZT48 solution, dilute it 2 times with ultrapure water, and detect the spectrum of the ZT48 self-assembled solution between 190 and 280 nm by circular dichroism spectroscopy.

[0114] The circular dichroism spectrum of the ZT48 self-assembled solution is as Figure 5 shown. The secondary structure of the ZT48 self-assembled solution has random coils, α-helices, β-turns and β-sheets. Among them, β-sheets and random coils are the main ones, and the proportion of the secondary structure is analyzed by CDpro software, and the results are shown in Table 2.

[0115] Table 2 Self-assembled secondary structure of ZT48

[0116] α - helix β - sheet β - turn Random coil 2.2％ 46.1％ 20.0％ 31.7％

[0117] Example 6

[0118] Detection of the effect of ZT48 on cell proliferation:

[0119] Using the CCK8 cell proliferation detection kit from Beyotime Biotechnology Company, detect the effect on cell proliferation. The specific operation is as follows: Inoculate mouse fibroblasts (L929 cells) in a 96-well plate at a density of 5000 cells per well, and incubate at 37 °C, 5% CO 2Cultivate overnight in an incubator. After it adheres completely, add 100 μl of complete medium containing ZT48 prepared in Example 1 per well. The concentration of ZT48 is 25, 50, and 100 ppm. After continuing to incubate in the incubator for 48 hours, add 10 μl of CCK-8 solution. After incubating in the incubator for 1 - 4 hours, measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the survival rate of L929 cells.

[0120] The detection results of the effect of ZT48 on cell proliferation are shown in Figure 6 As shown, in the concentration range of 25 - 100 ppm, ZT48 can significantly promote the proliferation of HaCaT cells. At 100 ppm, it can promote cell proliferation by 26.6%.

[0121] Example 7

[0122] Detection of the effect of ZT48 on cell migration:

[0123] Take keratinocytes (HaCaT) in the logarithmic growth phase and inoculate them into a 12-well plate at a density of 1×10 6 cells / well. Incubate overnight at 37°C in 5% CO 2 until they adhere completely and grow to full confluence. Scratch the cells in the 12-well plate and take a photo to record the cell growth state at 0 h. Add serum-free medium containing ZT48 prepared in Example 1 to the 12-well plate, 1 ml per well, so that the final concentration of ZT48 is 25 ppm, 50 ppm, 100 ppm. Use the group without any drug treatment as the blank control group. Incubate at 37°C in 5% CO 2 for 24 h, then take a photo to record the cell growth state at 24 h. Calculate the cell migration rate of the treatment group relative to the blank control group based on the area of the scratched area.

[0124] The detection results of the effect of ZT48 on cell migration are as shown in Figure 7 As shown, at concentrations of 50 and 100 ppm, ZT48 can significantly promote cell migration. At 100 ppm, it can promote cell migration by 26%.

[0125] Example 8

[0126] Detection of the effect of ZT48 on cell adhesion:

[0127] Add 0.5 ml of the ZT48 solution prepared in Example 1 to untreated 24-well plates (where cells cannot adhere normally) (using PBS solution as the blank control group). The concentration of ZT48 is 100 ppm, 200 ppm, and 400 ppm. Incubate and coat overnight at 4°C, remove the coating solution, and air-dry the residual liquid in a 37°C incubator before use.

[0128] Take keratinocytes (HaCaT) in the logarithmic growth phase and seed them in a 24-well plate at a density of 10×10 4 cells / well. Incubate at 37°C and 5% CO 2 for 24 h. Aspirate the medium, add 200 μl of Calcein AM staining working solution to each well for staining. Viable cells are stained with green fluorescence, and the cell adhesion status is observed by taking pictures with a fluorescence microscope.

[0129] The detection results of ZT48 on cell adhesion are as Figure 8 shown. The cells in the blank control group did not adhere normally, the cell morphology was round, and almost all the cells died. The cells in the ZT48 experimental group showed a normal adherent growth state, and the effect was best at 400 ppm. It shows that the self-assembled solution of ZT48 can promote cell adhesion. This may be because the network structure formed by the self-assembled short peptide ZT48 provides a scaffold environment for cell adhesion, enabling the cells to adhere and grow normally.

[0130] Example 9

[0131] Detection of ZT48 on the repair of keratinocyte barrier:

[0132] Take keratinocytes (HaCaT) in the logarithmic growth phase and seed them in a 12-well plate at a density of 5×10 5 cells / well. Incubate at 37°C and 5% CO 2 overnight until complete adhesion, and culture until about 60% confluence. Add the serum-free medium containing ZT48 prepared in Example 1 to the 12-well plate, 1 ml per well, so that the final concentration of ZT48 is 100 ppm and 200 ppm. The group without any drug treatment is used as the blank control group. Incubate at 37°C and 5% CO 2 for 24 h, then collect the cells and extract total RNA using an RNA extraction kit, reverse transcribe it into cDNA, and detect the relative expression levels of keratinocyte barrier repair-related genes, including keratin 1 (KRT-1), keratin 5 (KRT-5), filaggrin (FLG-1), and aquaporin (AQP3) by qPCR (fluorescence quantitative).

[0133] The results of ZT48 on the repair of keratinocyte barrier are as Figure 9 shown. Compared with the blank control group, ZT48 can significantly promote the expression of genes related to the repair of the keratinocyte skin barrier, and the effect is better at 100 ppm. At 100 ppm, it can promote the high expression of KRT-1 by 62.7%, the high expression of KRT-5 by 51.4%, the high expression of FLG-1 by 33.6%, and the high expression of AQP3 by 39.7%. It can be seen that the self-assembled short peptide has a strong function of repairing the skin barrier.

[0134] In summary, the present invention provides a multifunctional self-assembling short peptide ZT48, its preparation method and application, and a short peptide ZT48 self-assembly. The multifunctional self-assembling short peptide ZT48 fixes tyrosine as the tail and connects phenylalanine and tryptophan as the head, providing a benzene ring structure for the entire molecular structure, enabling it to self-assemble through π-π stacking interactions; the 5 amino acids in the middle of the molecule have their residues R 1 、R 2 、R 3 、R 4 and R 5 represented, R 1 ~R 5 are alternately connected by hydrophobic and hydrophilic amino acids. The hydrophilic residues provide hydrophilicity for the short peptide molecule and can form hydrogen bonds with water molecules. At the same time, in the polypeptide molecule, hydrogen bonds can be formed between the free amino group, imino group and hydroxyl group on the amino acid residue and the oxygen atom on the carbonyl group to regulate the self-assembly of the polypeptide molecule. The hydrophobic residues provide hydrophobicity for the short peptide molecule, and through hydrophobic interactions, the molecules are arranged orderly to regulate the self-assembly behavior of the polypeptide molecule; in addition, among the 5 amino acids in the middle of the molecule, two hydrophilic residues carry opposite charges, providing electrostatic interaction for the short peptide molecule, enabling it to self-assemble through electrostatic interaction. In addition, the nano-network self-assembly formed by the multifunctional self-assembling short peptide ZT48 through a hydrogen bond network can provide a scaffold for cells, promote cell adhesion, simulate the extracellular matrix conditions, thereby promoting cell proliferation and migration, and promoting the repair of the cell barrier.

[0135] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. A multifunctional self-assembling short peptide ZT48, characterized in that: Its chemical structure is shown below: Among them, R1, R3, and R5 in the formula are hydrophobic residues, and R2 and R4 are hydrophilic residues.

2. The multifunctional self-assembling short peptide ZT48 according to claim 1, characterized in that: The amino acids where the R1, the R3 and the R5 are located are independently selected from one of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline and tryptophan.

3. The multifunctional self-assembling short peptide ZT48 according to claim 1, characterized in that: The amino acids where the R2 and the R4 are located are independently selected from one of arginine, lysine, histidine, aspartic acid, and glutamic acid; and the R2 and the R4 have opposite charges.

4. The multifunctional self-assembling short peptide ZT48 according to claim 1, characterized in that: The chemical structural formula of the multifunctional self-assembling short peptide ZT48 is:

5. A method for preparing the multifunctional self-assembling short peptide ZT48 according to any one of claims 1 to 4, characterized in that: Includes steps: Using an eluent to remove Fmoc from Fmoc-Phe-Wang Resin, and obtaining a first solid phase substance after solid-liquid separation; After the first solid phase material and Fmoc-Trp(Boc)-OH are mixed and reacted, Fmoc is removed by using an eluent, and after solid-liquid separation, a second solid phase material is obtained; An amino acid containing R1 is added to the second solid phase material to react, and after the reaction, Fmoc removal is performed using an eluent, and then amino acids containing R2-R5 are used to complete the reaction and remove Fmoc in sequence, and after solid-liquid separation, a third solid phase material is obtained; After the third solid phase material is mixed with Fmoc-Tyr(tBu)-OH for reaction, Fmoc is removed by using an eluent, and after solid-liquid separation, a fourth solid phase material is obtained; Using acetic anhydride to perform N-terminal acetylation on the fourth solid phase material to obtain a resin; The resin and the cleavage solution are subjected to a cleavage reaction to obtain a reaction residue, and the reaction residue is precipitated using glacial isopropyl ether to obtain a multifunctional self-assembling short peptide ZT48.

6. The method for preparing the multifunctional self-assembling short peptide ZT48 according to claim 5, characterized in that: The eluent is composed of diethylamine dissolved in N,N-dimethylformamide; the concentration of the eluent is 5wt%-30wt%.

7. The method for preparing the multifunctional self-assembling short peptide ZT48 according to claim 5, characterized in that: After the solid-liquid separation step, a washing process is included; the solvent used in the washing process includes one or more of methanol, ethanol, DMF, and DCM.

8. The method for preparing the multifunctional self-assembling short peptide ZT48 according to claim 5, characterized in that: The cleavage solution is one or more of TFA, H2O, Tis, and DODT; and the cleavage reaction time is 1 h-3 h.

9. Use of the multifunctional self-assembling short peptide ZT48 as claimed in any one of claims 1 to 4 in the preparation of cosmetics, biomedical nanomaterials, tissue engineering materials, and drug delivery carriers.

10. A short peptide ZT48 self-assembly, characterized in that: The multifunctional self-assembling short peptide ZT48 according to any one of claims 1 to 4 is obtained through intramolecular or intermolecular self-assembly.

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