Co-assembly of self-assembled oligopeptide and recombinant III-type collagen as well as preparation method and application of co-assembly

Abstract

The invention discloses a co-assembly of self-assembled oligopeptide and recombinant III-type collagen as well as a preparation method and application of the co-assembly, and relates to the technical field of biomedical materials. The co-assembly body of the self-assembled oligopeptide and the recombinant III type collagen is obtained by co-assembling the self-assembled oligopeptide and the recombinant III type collagen; the self-assembled oligopeptide comprises hydrophobic amino acid, hydrophilic amino acid and charged amino acid, and the hydrophobic amino acid, the hydrophilic amino acid and the charged amino acid are connected through amide. According to the invention, the self-assembled oligopeptide and the recombinant III-type collagen are co-assembled to form a co-assembly body, a new assembled network structure is formed, and the self-assembled oligopeptide and the recombinant III-type collagen are mutually combined, so that the stability of the recombinant III-type collagen is enhanced, and the enzymolysis of the recombinant III-type collagen is inhibited. Meanwhile, the co-assembly body synergistically exerts the effects of the self-assembled oligopeptide and the recombinant III-type collagen, so that the effect of the recombinant III-type collagen can be further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a co-assembly of a self-assembling short peptide and recombinant type III collagen, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen has been widely used in the field of wound repair due to its good biocompatibility, low immunogenicity, and degradability. Animal collagen has the risks of immune response and disease transmission. With the development of gene recombination technology, the application of recombinant collagen for wound repair has become a research hotspot. The repeating unit of the amino acid sequence of recombinant type III collagen is the same as the specific functional region of the amino acid sequence of human collagen, and the functional region has a flexible triple helix structure of 164.88°, with good biocompatibility and high safety. However, recombinant type III collagen is easily enzymatically degraded, has poor stability, and its efficacy still needs to be further improved.

[0003] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0004] Based on the above deficiencies of the existing technology, the purpose of the present invention is to provide a co-assembly of a self-assembling short peptide and recombinant type III collagen, a preparation method thereof, and an application thereof, aiming to solve the problems that the existing recombinant type III collagen is easily enzymatically degraded, has poor stability, and its efficacy still needs to be further improved.

[0005] The technical solution of the present invention is as follows:

[0006] In the first aspect of the present invention, a co-assembly of a self-assembling short peptide and recombinant type III collagen is provided, wherein the co-assembly of the self-assembling short peptide and recombinant type III collagen is obtained by co-assembling the self-assembling short peptide and recombinant type III collagen; the self-assembling short peptide includes hydrophobic amino acids, hydrophilic amino acids, and charged amino acids, and the hydrophobic amino acids, hydrophilic amino acids, and charged amino acids are connected by amides.

[0007] Optionally, the hydrophobic amino acids include at least one of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, and tryptophan.

[0008] Optionally, the hydrophilic amino acids include at least one of asparagine, glutamine, serine, threonine, tyrosine, arginine, lysine, histidine, aspartic acid, glutamic acid, and cysteine.

[0009] Optionally, the charged amino acids include at least one of arginine, lysine, histidine, aspartic acid, and glutamic acid.

[0010] Optionally, the self-assembling short peptide comprises at least one of K8, R8, K5, ZT48, and K3, wherein the amino acid sequence of K8 is as shown in SEQ ID NO: 1, the amino acid sequence of R8 is as shown in SEQ ID NO: 2, the amino acid sequence of K5 is as shown in SEQ ID NO: 3, the amino acid sequence of ZT48 is as shown in SEQ ID NO: 4, and the amino acid sequence of K3 is KIK.

[0011] Optionally, the mass ratio of the self-assembling short peptide to the recombinant type III collagen is 1:(1 - 50).

[0012] In a second aspect of the present invention, there is provided a method for preparing a co-assembly of a self-assembling short peptide and recombinant type III collagen, which comprises the following steps:

[0013] Provide a first solution containing a self-assembling short peptide, the self-assembling short peptide comprising hydrophobic amino acids, hydrophilic amino acids, and charged amino acids, and the hydrophobic amino acids, hydrophilic amino acids, and charged amino acids being linked by amides;

[0014] Provide a second solution containing recombinant type III collagen;

[0015] After mixing the first solution and the second solution, perform ultrasonic treatment, and after standing for a preset time at a preset temperature, the self-assembling short peptide and the recombinant type III collagen undergo co-assembly to obtain the co-assembly of the self-assembling short peptide and the recombinant type III collagen.

[0016] Optionally, in the first solution, the concentration of the self-assembling short peptide is 0.01 - 10 mg / mL; in the second solution, the concentration of the recombinant type III collagen is 0.01 - 10 mg / mL;

[0017] The solvents in the first solution and the second solution each independently comprise at least one of water, physiological saline, and phosphate buffer solution.

[0018] Optionally, the power of the ultrasonic treatment is 100 - 600 W, the time of the ultrasonic treatment is 0 - 30 min, and the time of the ultrasonic treatment does not take 0 min; and / or,

[0019] The preset temperature is 25 - 45 °C, and the preset time is 1 - 7 days.

[0020] In the third aspect of the present invention, there is provided an application of a co-assembly of the self-assembling short peptide and recombinant type III collagen as described above in the present invention and / or a co-assembly of the self-assembling short peptide and recombinant type III collagen prepared by the preparation method as described above in the present invention in one or more fields of promoting cell repair, promoting cell adhesion, promoting cell proliferation, promoting collagen production, firming and anti-aging.

[0021] Beneficial effects: In the present invention, the self-assembling short peptide and recombinant type III collagen undergo co-assembly to form a co-assembly, forming a new assembled network structure. The self-assembling short peptide and recombinant type III collagen bind to each other, enhancing the stability of recombinant type III collagen and inhibiting the enzymatic hydrolysis of recombinant type III collagen. At the same time, the co-assembly synergistically exerts the effects of the self-assembling short peptide and recombinant type III collagen, and can further enhance the efficacy of recombinant type III collagen. Description of the Drawings

[0022] Figure 1 It is the mass spectrometry diagram of ZT48 in Example 1.

[0023] Figure 2 It is the circular dichroism spectrum diagram of ZT48 in Example 1.

[0024] Figure 3 It is the transmission electron microscopy diagram of the co-assembly in Example 1.

[0025] Figure 4 It is the transmission electron microscopy diagram of the co-assembly in Example 2.

[0026] Figure 5 It is the transmission electron microscopy diagram of the co-assembly in Example 3.

[0027] Figure 6 It is the transmission electron microscopy diagram of the co-assembly in Example 4.

[0028] Figure 7 It is the transmission electron microscopy diagram of ZT48 in Comparative Example 1.

[0029] Figure 8 It is the transmission electron microscopy diagram of recombinant type III collagen in Comparative Example 2.

[0030] Figure 9 It is the test result diagram of the performance of inhibiting the enzymatic hydrolysis of recombinant type III collagen.

[0031] Figure 10 It is the test result diagram of cell migration. Among them, (a) is the control diagram before and after cell migration, and (b) is the column diagram of relative migration rate.

[0032] Figure 11 It is the test result diagram of cell proliferation.

[0033] Figure 12 This is a graph showing the results of cell adhesion tests. Among them, (a) is a fluorescence image, and (b) is a bar graph of fluorescence intensity. Detailed implementation

[0034] The present invention provides a co-assembly of a self-assembling short peptide and recombinant type III collagen, as well as a preparation method and application thereof. To make the objectives, technical solutions, and effects of the present invention clearer and more definite, the present invention is 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.

[0035] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0036] If the description in the embodiments of the present invention involves "first", "second", etc., such descriptions of "first", "second", etc. are only for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the quantity of the indicated technical features.

[0037] The embodiments of the present invention provide a co-assembly of a self-assembling short peptide and recombinant type III collagen. Among them, the co-assembly of the self-assembling short peptide and recombinant type III collagen is obtained by co-assembling the self-assembling short peptide and recombinant type III collagen; the self-assembling short peptide includes hydrophobic amino acids, hydrophilic amino acids, and charged amino acids, and the hydrophobic amino acids, hydrophilic amino acids, and charged amino acids are connected by amide bonds.

[0038] Polypeptides have been widely used in the field of biomedical materials due to their good biocompatibility, low toxicity, and other advantages. With the rapid development of polypeptide self-assembly technology and polypeptide nanomaterials, in view of the deficiencies of polypeptide molecules, a variety of novel self-assembling nano-polypeptides have been designed and prepared successively, and have shown significant effects in the field of biomedical materials. The polypeptide self-assembly technology can self-assemble into a variety of nanostructures, including nanostructures such as nanoparticles, nanofibers, nanotubes, and nanonetworks, and co-assemble with ligand molecules through non-covalent bond forces to form co-assembled nanomaterials.

[0039] Specifically, in the present invention, hydrophobic amino acids, hydrophilic amino acids, and charged amino acids (specifically, positively charged amino acids or negatively charged amino acids) are the key for the short peptide to have self-assembly performance. The hydrophobic amino acids, hydrophilic amino acids, and charged amino acids are connected by amide bonds to form a polypeptide chain (the connection process and conditions are not particularly limited, and the conventional connection processes and conditions in the art can be used). In addition, the amino acid residues undergo self-assembly through non-covalent intermolecular forces (including hydrophilicity-hydrophobicity, hydrogen bonds, electrostatic forces, or π-π stacking, etc.) to form a nanostructure. Among them, hydrophobic amino acids can enhance the hydrophobicity of the molecule in the self-assembling short peptide molecule, and hydrophilic amino acids can enhance the hydrophilicity of the molecule in the self-assembling short peptide molecule, so as to achieve the balance of hydrophilicity-hydrophobicity of the molecule. Charged amino acids endow the molecule with a charge property in the self-assembling short peptide molecule, playing a role in enhancing the stability of the self-assembling short peptide. When the charged amino acid is a positively charged amino acid, the negatively charged cell membrane surface site can have an electrostatic interaction with the self-assembling short peptide, attracting the self-assembling short peptide and promoting the effective uptake of the cell.

[0040] In the present invention, the self-assembling short peptide and recombinant type III collagen are co-assembled to form a co-assembly, forming a new assembled network structure. The self-assembling short peptide and recombinant type III collagen are combined with each other, enhancing the stability of recombinant type III collagen and inhibiting the enzymatic hydrolysis of recombinant type III collagen. At the same time, the co-assembly synergistically exerts the effects of the self-assembling short peptide and recombinant type III collagen. Specifically, the co-assembly of the present invention reaches inside the cell by virtue of the cell-penetrating effect of the self-assembling short peptide, carrying the large-molecular-weight recombinant type III collagen into the cell interior, and exerting its functions of promoting cell proliferation, promoting cell migration, and promoting cell adhesion. The co-assembly can enhance the efficacy of recombinant type III collagen and play a synergistic role in tissue repair.

[0041] In the examples of the present invention, all recombinant type III collagens described in the prior art can be used, and the repeating unit of the amino acid sequence of recombinant type III collagen is the same as the specific functional region of the human collagen amino acid sequence. The functional domain is connected by the "g-x-y" amino acid triplet, where g is glycine, x is any amino acid, and y is proline.

[0042] In some embodiments, the hydrophobic amino acids include at least 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), and tryptophan (Trp, W).

[0043] In some embodiments, the hydrophilic amino acids include at least one of asparagine (Asn, N), glutamine (Gln, Q), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), arginine (Arg, R), lysine (Lys, K), histidine (His, H), aspartic acid (Asp, D), glutamic acid (Glu, E), and cysteine (Cys, C).

[0044] In some embodiments, the charged amino acids include at least one of arginine (Arg, R), lysine (Lys, K), histidine (His, H), aspartic acid (Asp, D), and glutamic acid (Glu, E).

[0045] In some embodiments, the self-assembling short peptides include at least one of K8, R8, K5, ZT48, and K3, wherein the amino acid sequence of K8 is as shown in SEQ ID NO: 1 (specifically KKIIIIKK), the amino acid sequence of R8 is as shown in SEQ ID NO: 2 (specifically RRIIIIRR), the amino acid sequence of K5 is as shown in SEQ ID NO: 3 (specifically KKIKK), the amino acid sequence of ZT48 is as shown in SEQ ID NO: 4 (specifically YPKIELWF), and the amino acid sequence of K3 is KIK.

[0046] In some embodiments, the mass ratio of the self-assembling short peptide to the recombinant type III collagen is 1:(1 - 50). By way of example, the mass ratio of the self-assembling short peptide to the recombinant type III collagen is 1:1, 1:2, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, etc.

[0047] The embodiment of the present invention also provides a method for preparing a co-assembly of a self-assembling short peptide and recombinant type III collagen, which includes the following steps:

[0048] S1. Provide a first solution containing a self-assembling short peptide, the self-assembling short peptide includes hydrophobic amino acids, hydrophilic amino acids, and charged amino acids, and the hydrophobic amino acids, hydrophilic amino acids, and charged amino acids are connected by amide bonds;

[0049] S2. Provide a second solution containing recombinant type III collagen;

[0050] S3. After mixing the first solution and the second solution, perform ultrasonic treatment, and after standing for a preset time at a preset temperature, the self-assembling short peptide and the recombinant type III collagen undergo co-assembly to obtain the co-assembly of the self-assembling short peptide and the recombinant type III collagen.

[0051] The preparation method of the present invention is simple and has a relatively low cost. The co-assembled body obtained can inhibit the enzymatic hydrolysis of recombinant type III collagen, improve the stability of recombinant type III collagen, and at the same time, the co-assembled body obtained can promote cell repair, cell adhesion, cell proliferation, and collagen production.

[0052] In this embodiment, ultrasonic treatment is used to assist the complete dissolution of the self-assembling peptide and recombinant type III collagen, disrupt the molecular arrangement, and make the molecules evenly dispersed, providing preparatory conditions for subsequent co-assembly. Since co-assembly is the connection of molecules by weak intermolecular forces, the purpose of standing is to avoid the disturbance of the autonomous movement of molecules by external forces. Under standing conditions, molecules can fully assemble with each other, and the formed stable co-assembled body will not disintegrate due to external forces.

[0053] In step S1, for the preparation of the self-assembling peptide, there are no special limitations on the connection process and conditions, and the conventional connection process and conditions in the art can be used.

[0054] In some embodiments, in the first solution, the concentration of the self-assembling peptide is 0.01 - 10 mg / mL, for example, it can be 0.01 mg / mL, 0.05 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL, etc. In a further embodiment, in the first solution, the concentration of the self-assembling peptide is 0.1 - 5 mg / mL.

[0055] In some embodiments, the solvent in the first solution includes at least one of water (preferably ultrapure water), physiological saline, and phosphate buffer solution.

[0056] In step S2, in some embodiments, in the second solution, the concentration of recombinant type III collagen is 0.01 - 10 mg / mL, for example, it can be 0.01 mg / mL, 0.05 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL, etc. In a further embodiment, in the second solution, the concentration of recombinant type III collagen is 0.1 - 5 mg / mL.

[0057] In some embodiments, the solvent in the second solution includes at least one of water (preferably ultrapure water), physiological saline, and phosphate buffer solution.

[0058] In step S3, during the co-assembly process of the self-assembling short peptide and recombinant type III collagen, the self-assembling short peptide can bind to recombinant type III collagen (composed of amino acids) through hydrogen bonding to undergo co-assembly. In addition, the self-assembling short peptide (nanostructure) can also be embedded with recombinant type III collagen, or the self-assembly structure of the self-assembling short peptide can wrap recombinant type III collagen.

[0059] In some embodiments, the power of the ultrasonic treatment is 100 - 600 W, for example, it can be 100 W, 200 W, 300 W, 400 W, 500 W or 600 W, etc.; the time of the ultrasonic treatment is 0 - 30 min, and the time of the ultrasonic treatment does not take 0 min, for example, the time of the ultrasonic treatment is 1 min, 2 min, 5 min, 10 min, 15 min, 20 min, 25 min or 30 min, etc.

[0060] In some embodiments, the preset temperature is 25 - 45 °C, for example, it can be 25 °C, 30 °C, 35 °C, 40 °C or 45 °C, etc., and the preset time is 1 - 7 days, for example, it can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, etc.

[0061] The embodiments of the present invention also provide an application of the co-assembly of the self-assembling short peptide and recombinant type III collagen as described above in the present invention and / or the co-assembly of the self-assembling short peptide and recombinant type III collagen prepared by the preparation method as described above in the present invention in one or more fields of promoting cell repair, promoting cell adhesion, promoting cell proliferation, promoting collagen production, firming and anti-aging.

[0062] The present invention will be further described below through specific examples.

[0063] Example 1

[0064] ZT48 (i.e., the self-assembling short peptide with the amino acid sequence of YPKIELWF) was prepared by the solid-phase synthesis method protected by Fmoc (fluorenylmethyloxycarbonyl), specifically as follows:

[0065] (1) Coupling of the first Phe to Wang resin

[0066] Weigh an appropriate amount of Wang resin and place it in a polypeptide synthesis column, add 200 mL of DCM (dichloromethane) to swell for 35 min, and drain the solvent.

[0067] Weigh 31.48 g of Fmoc-Phe-OH (fluorenylmethyloxycarbonyl-phenylalanine) and 10.98 g of HOBT (1-hydroxybenzotriazole), dissolve them in 100 mL of DMF (N,N-dimethylformamide), pre-activate at -20 °C for 25 min, add 10.26 g of DIC (N,N'-diisopropylcarbodiimide) and 1 g of DMAP (4-dimethylaminopyridine), stir to dissolve, add to the peptide synthesis column and react for 3 h. After the reaction is completed, drain the solvent, wash twice with DMF and once with DCM. Then, add 150 mL of a mixed solution containing propionic anhydride (2.54 g), DMAP (0.24 g), and NMM (i.e., N-methylmorpholine, 0.66 g), react for 1 h, drain the solvent, wash twice with DMF and once with DCM. Add 150 mL of a mixed solution of diethylamine and DMF (the volume content of diethylamine is 20%), react for 40 min, drain the solvent, wash once with DMF and once with DCM respectively, and the ninhydrin test shows positive.

[0068] (2) Coupling of the second Trp

[0069] Add 6.85 g of Fmoc-Trp(Boc)-OH (fluorenylmethyloxycarbonyl-tryptophan-ε-tert-butoxycarbonyl), 1.76 g of HOBT, 1.64 g of DIC and 100 mL of DMF to the reactor, react for 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. Then add 150 mL of a mixed solution of diethylamine and DMF (the volume content of diethylamine is 20%), react for 40 min, drain the solvent, wash once with DMF and once with DCM respectively, and the ninhydrin test shows positive.

[0070] (3) Coupling of the third to the eighth amino acids

[0071] Refer to step (2), couple Fmoc-Leu-OH (fluorenylmethyloxycarbonyl-leucine), Fmoc-Glu(OtBu)-OH (fluorenylmethoxycarbonyl-glutamic acid-1-tert-butyl ester), Fmoc-Ile-OH (fluorenylmethyloxycarbonyl-isoleucine), Fmoc-Lys(Boc)-OH (fluorenylmethyloxycarbonyl-lysine-ε-tert-butoxycarbonyl), Fmoc-Pro-OH (fluorenylmethyloxycarbonyl-proline), Fmoc-Tyr(tBu)-OH (fluorenylmethoxycarbonyl-tyrosine-oxy-tert-butyl) in sequence. After the coupling of the eighth amino acid is completed, add acetic anhydride to acetylate the -NH 2 at the N-terminus.

[0072] (4) After the reaction was completed, the solvent was pressed dry, and it was washed 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, once with 150 mL of methanol for shrinkage, and dried in vacuo at 39 °C overnight.

[0073] (5) Use 150 mL of cleavage solution (where trifluoroacetic acid:H 2 O:triisopropylsilane:2,2'-(1,2-ethylenedioxy)bis(ethanethiol) = 92.5:2.5:2.5:2.5, volume ratio) to cleave the above resin for 3 h. After concentrating and removing most of the trifluoroacetic acid with a rotary evaporator, it was added to 10 times the volume of ice-cold isopropyl ether for precipitation, and the solid crude peptide ZT48 was obtained by filtration.

[0074] (6) Weigh 500 mg of the crude peptide ZT48, dissolve and dilute it to 20 mL with the mobile phase. Filter it with a 0.45 μm organic filter membrane, take the subsequent filtrate, put it into a high-performance liquid chromatograph, and perform gradient separation with the high-performance liquid chromatograph. Collect the corresponding mobile phase at the retention time of the target absorption peak, and then obtain the ZT48 solution. At 40 °C, concentrate and freeze-dry the collected qualified solution to obtain the white powdery solid ZT48 (purity: 95.4%).

[0075] Among them, the high-performance liquid chromatography conditions are as follows:

[0076] Chromatographic column: C18 chromatographic column (4.6×250 mm, 5 μm);

[0077] Mobile phase A: aqueous solution of trifluoroacetic acid (volume content of trifluoroacetic acid is 0.1%);

[0078] Mobile phase B: acetonitrile;

[0079] Flow rate: 1 mL / min;

[0080] Column temperature: 30 °C;

[0081] Detection wavelength: 250 nm, 210 nm;

[0082] Injection volume: 5 μL.

[0083] The high-performance liquid chromatography (HPLC) gradient analysis method is shown in Table 1.

[0084] Table 1. HPLC gradient analysis method

[0085] Experiment time Mobile phase A (volume content) Mobile phase B (volume content) 0 - 5 min 95％ 5％ Equilibration 5 - 30 min 5％ 95％ Gradient 30 - 40 min 95％ 5％ Equilibration 40 - 45 min 95％ 5％ Equilibration

[0086] The mass spectrum of ZT48 is as Figure 1 shown, indicating that the synthesis of ZT48 was successful.

[0087] The circular dichroism spectrum of ZT48 is as follows Figure 2 shown (where mdeg is millidegree). The secondary structure of ZT48 includes random coils, α - helices, β - turns and β - sheets. Among them, it is mainly composed of β - sheets and random coils. The proportion of the secondary structure is analyzed by CDpro software, and the results are shown in Table 2.

[0088] Table 2. Self - assembled secondary structure of ZT48

[0089] α - helix β - sheet β - turn Random coil 2.2％ 46.1％ 20.0％ 31.7％

[0090] This example also provides a preparation method for the co - assembly of a self - assembling short peptide and recombinant type III collagen, including the following steps:

[0091] Mix ZT48 prepared by the above method with ultrapure water to prepare a first solution with a concentration of 2 mg / mL of the self - assembling short peptide ZT48;

[0092] Mix recombinant type III collagen (purchased from Juyuan Biotechnology Co., Ltd., Zhuji, Zhejiang, product model SFH01) with ultrapure water to prepare a second solution with a concentration of 5 mg / mL of recombinant type III collagen;

[0093] Mix equal volumes of the first solution and the second solution, ultrasonicate at 200 W for 10 min, and then transfer to stand at 37 °C for 3 days to obtain a solution containing the co - assembly of ZT48 and recombinant type III collagen (abbreviated as co - assembly).

[0094] Prepare a transmission electron microscopy sample and conduct transmission electron microscopy testing: Take 10 μL of the solution containing the co - assembly prepared in Example 1, dilute it 5 times with ultrapure water, drop it on a carbon - supported copper grid (200 mesh), after standing and sedimenting for 30 min, use a disposable filter paper to suck away the liquid drop, air - dry overnight, and observe and photograph using a biological transmission electron microscope.

[0095] The transmission electron microscopy image of the co - assembly is as follows Figure 3 shown. It can be seen that recombinant type III collagen is embedded in the self - assembled network of ZT48, and the co - assembly mainly has the morphology of a self - assembled network structure dominated by ZT48.

[0096] Example 2

[0097] This example provides a preparation method for the co - assembly of a self - assembling short peptide and recombinant type III collagen, and the steps are as follows:

[0098] Entrust Shenzhen Readlin Biotechnology Co., Ltd. to prepare K8 (i.e., the self - assembling short peptide with the amino acid sequence KKIIIIKK) by solid - phase peptide synthesis method.

[0099] Mix K8 with ultrapure water to prepare a first solution with a K8 concentration of 2 mg / mL;

[0100] Mix recombinant type III collagen (purchased from Jiangsu Chuangjian Medical Technology Co., Ltd., product model TTA01 - 02SM1) with ultrapure water to prepare a second solution with a recombinant type III collagen concentration of 5 mg / mL;

[0101] Mix equal volumes of the first solution and the second solution, sonicate at 200 W for 10 min, and then transfer to a temperature of 37 °C and let stand for 3 days to obtain a solution containing the co - assembly of K8 and recombinant type III collagen (abbreviated as co - assembly).

[0102] Prepare a transmission electron microscopy sample and conduct a transmission electron microscopy test: Take 10 μL of the solution containing the co - assembly prepared in Example 1, 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 overnight, and observe and photograph using a biological transmission electron microscope.

[0103] The transmission electron micrograph of the co - assembly is as Figure 4 shown. It can be seen that K8 and recombinant type III collagen aggregate with each other, connect into chains, and the chains are interconnected with each other to form a network structure, constituting the co - assembly.

[0104] Example 3

[0105] This example provides a method for preparing a co - assembly of a self - assembling short peptide and recombinant type III collagen, including the following steps:

[0106] Entrust Shenzhen Readlin Biotechnology Co., Ltd. to prepare R8 (i.e., the self - assembling short peptide with the amino acid sequence RRIIIIRR) by solid - phase peptide synthesis.

[0107] Mix R8 with ultrapure water to prepare a first solution with an R8 concentration of 2 mg / mL;

[0108] Mix recombinant type III collagen (purchased from Jiangsu Chuangjian Medical Technology Co., Ltd., product model TTA01 - 02SM1) with ultrapure water to prepare a second solution with a recombinant type III collagen concentration of 5 mg / mL;

[0109] Mix equal volumes of the first solution and the second solution, sonicate at 200 W for 10 min, and then transfer to a temperature of 37 °C and let stand for 3 days to obtain a solution containing the co - assembly of R8 and recombinant type III collagen (abbreviated as co - assembly).

[0110] Preparation of transmission electron microscope (TEM) samples and TEM testing: Take 10 μL of the solution containing the co-assembly prepared in Example 2, dilute it 5 times with ultrapure water, drop it on a carbon-supported copper grid (200 mesh), after standing and sedimenting for 30 min, 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.

[0111] The TEM image of the co-assembly is as Figure 5 shown. It can be seen that R8 and recombinant type III collagen aggregate with each other to form nanoparticles, and the nanoparticles are interconnected with each other, presenting an irregular morphology, constituting the co-assembly.

[0112] Example 4

[0113] This example provides a preparation method of a co-assembly of a self-assembled short peptide and recombinant type III collagen, and the steps are as follows:

[0114] Mix K8 (its amino acid sequence and preparation method are the same as those in Example 2) and ultrapure water to prepare a first solution with a K8 concentration of 2 mg / mL;

[0115] Mix recombinant type III collagen (purchased from Zhejiang Zhuji Juyuan Biotechnology Co., Ltd., product model SFH01) and ultrapure water to prepare a second solution with a recombinant type III collagen concentration of 5 mg / mL;

[0116] Mix equal volumes of the first solution and the second solution, ultrasonicate it at 200 W for 10 min, and then transfer it to stand at 37 °C for 3 days to obtain a solution containing the co-assembly of K8 and recombinant type III collagen (abbreviated as co-assembly).

[0117] Preparation of transmission electron microscope (TEM) samples and TEM testing: Take 10 μL of the solution containing the co-assembly prepared in Example 3, dilute it 5 times with ultrapure water, drop it on a carbon-supported copper grid (200 mesh), after standing and sedimenting for 30 min, 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.

[0118] The TEM image of the co-assembly is as Figure 6 shown. It can be seen that K8 and recombinant type III collagen aggregate with each other to form larger-sized nanoparticles, constituting the co-assembly, and the distribution is uniform.

[0119] Example 5

[0120] This example provides a preparation method of a co-assembly of three self-assembled short peptides and recombinant type III collagen. The difference from Example 2 is only that:

[0121] The first type: In the first solution used, the concentration of K8 is 0.1 mg / mL, and the concentration of recombinant type III collagen in the second solution is 6.38 mg / mL, to obtain a solution containing co-assembly 1.

[0122] The second type: In the first solution used, the concentration of K8 is 1.0 mg / mL, and the concentration of recombinant type III collagen in the second solution is 7.30 mg / mL, to obtain a solution containing co-assembly 2.

[0123] The third type: In the first solution used, the concentration of K8 is 5.0 mg / mL, and the concentration of recombinant type III collagen in the second solution is 7.84 mg / mL, to obtain a solution containing co-assembly 3.

[0124] Comparative Example 1

[0125] This comparative example provides a method for preparing a self-assembled short peptide solution, including the following steps:

[0126] Mix ZT48 (its amino acid sequence and preparation method are the same as those in Example 1) with ultrapure water to prepare a first solution with a ZT48 concentration of 2 mg / mL;

[0127] Mix an equal volume of the first solution and ultrapure water, sonicate at 200 W for 10 min, and then transfer to stand at 37 °C for 3 days to obtain a ZT48 solution.

[0128] Prepare a transmission electron microscopy sample and conduct transmission electron microscopy testing: Take 10 μL of the ZT48 solution prepared in Comparative Example 1, dilute it 5 times with ultrapure water, drop it on a carbon-supported copper grid (200 mesh), after standing and settling for 30 min, use a disposable filter paper to suck away the liquid drop, air dry overnight, and observe and photograph using a biological transmission electron microscope.

[0129] The transmission electron microscopy image of ZT48 is as Figure 7 shown. It can be seen that the self-assembled short peptide of ZT48 presents an obvious network structure.

[0130] Comparative Example 2

[0131] This comparative example provides a method for preparing a recombinant type III collagen solution, including the following steps:

[0132] Mix recombinant type III collagen (purchased from Zhejiang Zhuji Juyuan Biotechnology Co., Ltd., product model SFH01) with ultrapure water to prepare a second solution with a recombinant type III collagen concentration of 5 mg / mL;

[0133] Mix an equal volume of the second solution and ultrapure water, sonicate at 200 W for 10 min, and then transfer to stand at 37 °C for 3 days to obtain a recombinant type III collagen solution.

[0134] Preparation of transmission electron microscopy (TEM) samples and TEM testing: Take 10 μL of the recombinant type III collagen solution prepared in Comparative Example 2, dilute it 5 times with ultrapure water, drop it on a carbon-supported copper grid (200 mesh), after standing and sedimenting for 30 min, use a disposable filter paper to suck away the liquid droplet, air dry it overnight naturally, and observe and photograph it using a biological transmission electron microscope.

[0135] The TEM image of the recombinant type III collagen is as Figure 8 shown. It can be seen that the recombinant type III collagen presents an obvious nanoparticle morphology.

[0136] Functional testing of the co-assembly:

[0137] (1) Test for inhibiting the enzymatic hydrolysis performance of recombinant type III collagen, specifically as follows:

[0138] Mix the recombinant type III collagen used in Example 5 with ultrapure water to prepare a second solution with a recombinant type III collagen concentration of 6.24 mg / mL.

[0139] Mix an equal volume of the second solution and PBS (phosphate buffer solution), sonicate it at 200 W for 10 min, and then transfer it to stand at 37 °C for 3 days to obtain a recombinant type III collagen solution, denoted as the control group.

[0140] Use the solutions containing co-assembly 1, co-assembly 2, and co-assembly 3 in Example 5 as the experimental groups.

[0141] Respectively test the concentration of recombinant type III collagen in the control group and experimental group solutions as the initial concentration. As Figure 9 shown, the concentration of recombinant type III collagen in the control group solution is 3.12 mg / mL. In the experimental groups, the concentrations of recombinant type III collagen in the solutions containing co-assembly 1, co-assembly 2, and co-assembly 3 are 3.19 mg / mL, 3.65 mg / mL, and 3.92 mg / mL, respectively.

[0142] Add 0.1 mg / mL of collagenase to the control group and experimental group solutions respectively, and enzymatically hydrolyze them at 37 °C for 30 min. After enzymatic hydrolysis, detect the concentration of recombinant type III collagen in each group, denoted as the concentration after enzymatic hydrolysis, and compare it with the initial concentration. As Figure 9 shown, after enzymatic hydrolysis, the concentration of recombinant type III collagen in the control group solution is 2.18 mg / mL; in the experimental groups, the concentrations of recombinant type III collagen in the solutions containing co-assembly 1, co-assembly 2, and co-assembly 3 are 2.54 mg / mL, 3.14 mg / mL, and 3.76 mg / mL, respectively.

[0143] It can be seen that in the control group, about 30% of the recombinant type III collagen that did not form co-assemblies without the addition of self-assembling short peptides was enzymatically hydrolyzed; the amount of recombinant type III collagen enzymatically hydrolyzed in the solution containing co-assemblies was significantly lower than that in the control group, and as the concentration of the self-assembling short peptides increased, the amount of recombinant type III collagen enzymatically hydrolyzed gradually decreased. The amount of recombinant type III collagen enzymatically hydrolyzed in the solution containing co-assembly 1 was 20%, the amount of recombinant type III collagen enzymatically hydrolyzed in the solution containing co-assembly 2 was 14%, and the amount of recombinant type III collagen enzymatically hydrolyzed in the solution containing co-assembly 3 was 4%. Therefore, the above results indicate that the co-assemblies can significantly inhibit the enzymatic hydrolysis of recombinant type III collagen and enhance its stability.

[0144] (2) Cell migration test, specifically as follows:

[0145] Keratinocytes (HaCaT) in the logarithmic growth phase were seeded in a 12-well plate at a density of 1×10 6 cells / well. Incubated overnight at 37°C and 5% CO 2 until completely adherent, and cultured until completely confluent. The cells in the 12-well plate were scratched, and the cell growth state at 0 h was photographed and recorded.

[0146] The co-assemblies, ZT48 solution, and recombinant type III collagen solution prepared in Example 1, Comparative Example 1, and Comparative Example 2 were added to a 12-well plate containing serum-free medium, 1 mL per well, so that the final concentration of the co-assemblies was 0.35 mg / mL, the final concentration of ZT48 was 0.1 mg / mL, and the final concentration of recombinant type III collagen was 0.25 mg / mL, as the treatment group. No drug was added as the blank control group. Incubated at 37°C and 5% CO 2 for 24 h, and the cell growth state at 24 h was photographed and recorded. The cell migration rate of the treatment group relative to the blank control group was calculated based on the scratched area.

[0147] The results are as Figure 10 shown, where *** indicates P<0.001, * indicates P<0.05, ns indicates no significant difference. It can be seen that the ability of recombinant type III collagen in Comparative Example 2 to promote the migration of HaCaT cells is weak, and ZT48 in Comparative Example 1 and the co-assemblies in Example 1 can significantly promote cell migration. The solution containing co-assemblies prepared in Example 1 has a stronger effect on promoting cell migration than the two monomeric molecule solutions in Comparative Example 1 and Comparative Example 2. This is because the formation of co-assemblies amplifies the advantages of single components, enabling the two single components to synergistically promote cell migration.

[0148] (3) Cell proliferation test, specifically as follows:

[0149] Take keratinocytes (HaCaT) in the logarithmic growth phase and seed them in a 96-well plate at a density of 3×10 3 cells / well. Incubate overnight at 37°C in 5% CO 2 until complete attachment. Add the co-assembly-containing solutions, ZT48 solution, and recombinant type III collagen solution prepared in Example 1, Comparative Example 1, and Comparative Example 2 respectively to the 96-well plate containing the culture medium (containing 5% serum), 0.1 mL per well, so that the final concentration of the co-assembly is 0.35 mg / mL, the final concentration of ZT48 is 0.1 mg / mL, and the final concentration of recombinant type III collagen is 0.25 mg / mL, as the treatment groups. Use the group without adding any drug as the blank control group. Incubate at 37°C in 5% CO 2 for 24 h, then add 10 μL of CCK-8 to each well, gently mix by oscillation, incubate in the incubator for 2 h, and measure the absorbance OD450 at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the cell survival rate of the treatment groups relative to the blank control group to evaluate the proliferation ability.

[0150] The results are as Figure 11 shown, where *** indicates P < 0.001 and * indicates P < 0.05. The ability of ZT48 in Comparative Example 1 and recombinant type III collagen in Comparative Example 2 to promote the proliferation of HaCaT cells is relatively weak, which can promote the proliferation of about 18% and 7% of the cells respectively; the co-assembly in Example 1 can significantly promote cell proliferation, and the cell proliferation rate is about 34%. The co-assembly-containing solution prepared in Example 1 has a stronger effect on promoting cell proliferation than the two monomeric molecule solutions of Comparative Example 1 and Comparative Example 2. This is because the formation of the co-assembly amplifies the advantages of the single components, enabling the two single components to synergistically promote cell proliferation.

[0151] (4) Cell adhesion test, specifically as follows:

[0152] Add 0.5 mL of the co-assembly-containing solutions, ZT48 solution, and recombinant type III collagen solution prepared in Example 1, Comparative Example 1, and Comparative Example 2 respectively to an untreated 24-well plate (where cells cannot adhere normally), use PBS solution as the blank control group, incubate and coat overnight at 4°C, remove the coating solution, and air-dry the residual liquid in a 37°C incubator for later use.

[0153] Take keratinocytes (HaCaT) in the logarithmic growth phase and seed them in the above 24-well plate at a density of 10×10 4 cells / well. Incubate at 37°C in 5% CO 2Incubate for 24 h under the conditions. Aspirate the culture medium, add 200 μL of Calcein AM staining working solution to each well for staining. Living cells are stained with green fluorescence. Observe the cell adhesion state by taking pictures with a microscope, and use the green fluorescence intensity as a measure of the number of adherent cells.

[0154] The results are as Figure 12 shown. Among them, *** indicates P < 0.001, and * indicates P < 0.05. It can be seen that in the well plates containing the recombinant type III collagen in Comparative Example 2 and the PBS solution (blank control group), the cells did not adhere normally and grew in clusters. The cell morphology was round and did not spread. In the well plates containing ZT48 in Comparative Example 1 and the co-assembly in Example 1, the cells grew in a normal adherent state. This shows that ZT48 in Comparative Example 1 and the co-assembly in Example 1 can promote cell adhesion. This is because the network structure formed by ZT48 provides a scaffold for cell adhesion. Calculated by fluorescence intensity, the number of adherent cells can show that the co-assembly in Example 1 has a more obvious effect on promoting cell adhesion than the self-assembled short peptide in Comparative Example 1, corresponding to the result of promoting cell proliferation.

[0155] The co-assembly of the self-assembled short peptide and recombinant type III collagen provided by the present invention has the effects of significantly promoting cell proliferation, promoting cell migration, and promoting cell adhesion, further proving its great application potential in the fields of biomedical materials and tissue injury repair materials.

[0156] 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 changes can be made according to the above description. All these improvements and changes should fall within the protection scope of the appended claims of the present invention.

Claims

1. A co-assembly of a self-assembling short peptide and recombinant type III collagen, characterized in that: The co-assembly of the self-assembling short peptide and recombinant type III collagen is obtained by co-assembling the self-assembling short peptide and recombinant type III collagen; the self-assembling short peptide includes hydrophobic amino acids, hydrophilic amino acids and charged amino acids, and the hydrophobic amino acids, hydrophilic amino acids and charged amino acids are connected by amides.

2. The co-assembly of the self-assembling short peptide and recombinant type III collagen according to claim 1, characterized in that: The hydrophobic amino acids include at least one of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline and tryptophan.

3. The co-assembly of the self-assembling short peptide and recombinant type III collagen according to claim 1, characterized in that: The hydrophilic amino acids include at least one of asparagine, glutamine, serine, threonine, tyrosine, arginine, lysine, histidine, aspartic acid, glutamic acid and cysteine.

4. The co-assembly of the self-assembling short peptide and recombinant type III collagen according to claim 1, characterized in that: The charged amino acids include at least one of arginine, lysine, histidine, aspartic acid and glutamic acid.

5. The co-assembly of the self-assembling short peptide and recombinant type III collagen according to claim 1, characterized in that: The self-assembling short peptide includes at least one of K8, R8, K5, ZT48 and K3, wherein the amino acid sequence of K8 is shown in SEQ ID NO: 1, the amino acid sequence of R8 is shown in SEQ ID NO: 2, the amino acid sequence of K5 is shown in SEQ ID NO: 3, the amino acid sequence of ZT48 is shown in SEQ ID NO: 4, and the amino acid sequence of K3 is KIK.

6. The co-assembly of the self-assembling short peptide and recombinant type III collagen according to claim 1, characterized in that: The mass ratio of the self-assembling short peptide to the recombinant type III collagen is 1:(1-50).

7. A method for preparing a co-assembly of a self-assembling short peptide and recombinant type III collagen, characterized in that: The steps include: Providing a first solution, wherein the first solution contains a self-assembling short peptide, wherein the self-assembling short peptide comprises a hydrophobic amino acid, a hydrophilic amino acid and a charged amino acid, wherein the hydrophobic amino acid, the hydrophilic amino acid and the charged amino acid are connected via amides; providing a second solution, wherein the second solution contains recombinant type III collagen; After the first solution and the second solution are mixed, ultrasonic treatment is performed, and after standing at a preset temperature for a preset time, the self-assembling short peptide and the recombinant type III collagen are co-assembled to obtain a co-assembly of the self-assembling short peptide and the recombinant type III collagen.

8. The preparation method according to claim 7, characterized in that: In the first solution, the concentration of the self-assembling short peptide is 0.01 to 10 mg / mL; in the second solution, the concentration of the recombinant type III collagen is 0.01 to 10 mg / mL; The solvents in the first solution and the second solution each independently include at least one of water, physiological saline and phosphate buffer solution.

9. The preparation method according to claim 7, characterized in that: The power of the ultrasonic treatment is 100-600W, the time of the ultrasonic treatment is 0-30min, and the time of the ultrasonic treatment is not 0min; and / or, The preset temperature is 25 to 45° C., and the preset time is 1 to 7 days.

10. Use of a co-assembly of a self-assembling short peptide according to any one of claims 1 to 6 and recombinant type III collagen and / or a co-assembly of a self-assembling short peptide and recombinant type III collagen prepared by the preparation method according to claims 7 to 9 in one or more of the fields of promoting cell repair, promoting cell adhesion, promoting cell proliferation, promoting collagen production, firming and anti-aging.

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