Preparation method and application of polypeptide molecule and piezoelectric polypeptide aggregate nanometer material

Abstract

The invention belongs to the field of preparation of polypeptide nano-materials, and discloses a preparation method and application of a polypeptide molecule and a piezoelectric polypeptide aggregate nano-material. Firstly, a PFFRGD hexapeptide molecule is designed and synthesized on the basis of amyloid polypeptide, nano-materials of different structures are synthesized through regulation and control of self-assembly, and the nano-structure materials are non-toxic to cells, have a piezoelectric effect and can well achieve cell proliferation.

Description

Technical Field

[0001] The present invention belongs to the field of preparation of polypeptide nanomaterials, and particularly refers to nano-aggregates with piezoelectric effect synthesized through regulated self-assembly of amyloid polypeptides, and their application in the field of biology. Background Art

[0002] In recent years, the preparation of biological micro-nanomaterials by the precise hierarchical self-assembly of polypeptide molecules through a "bottom-up" approach has gradually become a research hotspot in the fields of biomaterials and biomedicine. Generally, polypeptides have a short half-life in the body. Due to the lack of sufficient stimulation of membrane surface receptors, single polypeptide molecules are often degraded by enzymes or quickly internalized into the cytoplasm, thus limiting their application in the biological field. Recent studies have shown that the aggregates formed after assembly can resist protease degradation, significantly enhance the biological stability of these polypeptides, prolong the half-life, and have better biological activity. While regulating the misfolding and aggregation of amyloid polypeptides, researchers have designed a variety of supramolecular nanomaterials based on the self-assembly of amyloid polypeptides and short peptides, and they have shown great application potential in various biological application fields such as tissue engineering, cancer treatment, biomimetic catalysis, and biological antibacterial.

[0003] Based on the assembly of amyloid short peptide FF, the present invention designs and synthesizes an A-type peptide formed by combining a PFF short peptide with piezoelectric properties with a cell membrane-targeting RGD short peptide. C -PFFRGD-NH2 hexapeptide molecule was then used to synthesize nanomaterials with piezoelectric effect in different structures by regulating self-assembly, and it was well applied in biology. Summary of the invention

[0004] Based on amyloid peptide, an A C -PFFRGD-NH2 hexapeptide molecule, and synthesized piezoelectric polypeptide aggregate nanomaterials with different structures by regulating self-assembly. The nanostructured material is non-toxic to cells and has piezoelectric effect, which can promote cell proliferation well.

[0005] A method for preparing a polypeptide molecule comprises the following steps:

[0006] (1) Resin pretreatment:

[0007] Weigh 4-[(2,4-dimethoxyphenyl)(fluorenylmethoxycarbonyl-amino)methyl]phenoxyacetamidomethyl resin, put it into a vertical reactor, add dichloromethane DCM and soak for 30-60 minutes; then add a 20% piperidine / N,N-dimethylformamide (DMF) solution by volume into the reactor using a wash bottle to completely soak the resin in the solution, react with nitrogen bubbling for 20-30 minutes, and then wash with DMF several times;

[0008] (2) The first amino acid is attached to the resin:

[0009] Dissolve the aspartic acid derivative Fmoc-Asp(OTBU)-OH containing the amine protecting group Fmoc in DMF, then add N,N'-diisopropylcarbodiimide DIC and shake well, add to the reactor of step (1), react with nitrogen bubbling for 120 to 150 minutes, wash the resin with DMF several times after the reaction, add a mixed solution of acetic anhydride and DCM to the reactor, and then add N,N'-diisopropylethylamine DIEA to react for 20 to 25 minutes;

[0010] (3) Washing

[0011] First, after the liquid in the reactor of step (2) is drained with a circulating water vacuum pump, DMF is added to the reactor with a washing bottle to completely immerse the resin in the solution, and nitrogen bubbling is performed for 30 to 40 seconds, and then the liquid in the reactor is drained with a circulating water vacuum pump for 30 to 40 seconds, which is repeated several times;

[0012] Next, a 20% piperidine / DMF solution in a volume ratio is added to the reactor using a wash bottle to completely soak the resin in the solution, and nitrogen is bubbled for reaction for 20 to 25 minutes to remove Fmoc; after the reaction is completed, the liquid in the reactor is drained with a circulating water vacuum pump, and DMF is added to the reactor using a wash bottle to completely soak the resin in the solution, and nitrogen is bubbled for washing for 30 to 40 seconds, and then the liquid in the reactor is drained with a circulating water vacuum pump, and the draining time is 30 to 40 seconds, which is repeated several times;

[0013] Finally, the resin is used to detect whether the Fmoc removal is successful, and the resin is colored, indicating that the Fmoc removal is successful; and the resin A after being drained is obtained;

[0014] (4) The second amino acid is attached to the resin:

[0015] The glycine derivative Fmoc-Gly-OH protected by the Fmoc group and 1-hydroxybenzotriazole HOBT were placed in a centrifuge tube in proportion and fully dissolved with DMF; N,N'-diisopropylcarbodiimide DIC was then added and mixed, and then added to the reactor, mixed with the resin A after being drained, and nitrogen bubbling was performed for condensation reaction for 1 to 1.5 hours;

[0016] Using resin detection to detect whether the amino acid is successfully connected, until the resin is colorless; washing the product using the washing method described in step (3), and draining to obtain resin B;

[0017] (5) The third, fourth, fifth, and sixth amino acids are sequentially connected to the resin:

[0018] Using the condensation method of step (4), the arginine derivative Fmoc-Arg(PBF)-OH containing the amine protecting group Fmoc, two phenylalanine derivatives Fmoc-Phe-OH, and the proline derivative Fmoc-Pro-OH are sequentially connected to the resin B, and after washing using the washing method described in step (3), the resin C is obtained after being dried;

[0019] (6) N-terminal acetylation modification of polypeptide chain:

[0020] Add acetic anhydride and DCM mixture into the reactor containing resin C to ensure that resin C is completely immersed, add DIEA and react for 20 to 30 minutes. After the reaction, wash the resin several times with DMF and methanol, and then dry it to obtain resin D.

[0021] (7) Cutting:

[0022] Prepare a cutting reagent, including 95 ml trifluoroacetic acid (TFA) + 1 mL water + 2 mL 1,2-ethanedithiol (EDT) + 2 mL tris(hydroxymethyl)aminomethane (Tis), and precool anhydrous ether at -20°C for more than 2 hours;

[0023] Add resin D to the cleavage reagent and cleave for 2 to 2.5 hours, then precipitate with ether;

[0024] The synthesized peptide was purified by high performance liquid chromatography (HPLC) to remove unreacted amino acids and other impurities; the purified peptide solution was freeze-dried to obtain the final peptide A. C -PFFRGD-NH2 powder.

[0025] In steps (1) and (2), the amount ratio of 4-[(2,4-dimethoxyphenyl)(fluorenylmethoxycarbonyl-amino)methyl]phenoxyacetamidomethyl resin, aspartic acid derivative Fmoc-Asp(OTBU)-OH containing amine protecting group Fmoc, N,N'-diisopropylcarbodiimide (DIC) and N,N-diisopropylethylamine (DIEA) is 1 g: 0.3 mmol: 0.3 mmol: 0.3 mmol.

[0026] In steps (1) and (4), the ratio of 4-[(2,4-dimethoxyphenyl)(fluorenylmethoxycarbonyl-amino)methyl]phenoxyacetamidomethyl resin, Fmoc-Gly-OH, 1-hydroxybenzotriazole HOBT and N,N'-diisopropylcarbodiimide DIC is 1 g: 1-1.5 mmol: 1 mmol: 1 mmol.

[0027] The ratio of 4-[(2,4-dimethoxyphenyl)(fluorenylmethoxycarbonyl-amino)methyl]phenoxyacetamidomethyl resin in step (1) and DIEA in step (6) is 1 g:0.3 mmol.

[0028] In steps (2) and (6), the volume ratio of acetic anhydride to DCM in the mixed solution of acetic anhydride and DCM is 1:5.

[0029] A method for preparing a piezoelectric polypeptide aggregate nanomaterial by regulating polypeptide A C -PFFRGD-NH2 self-assembles to form nanomaterials with different structures and piezoelectric properties. The specific steps are:

[0030] Peptide A C -PFFRGD-NH2 is dissolved in a PBS solution or deionized water filtered through a sterile filter membrane, and then incubated in a constant temperature oscillator to obtain a piezoelectric polypeptide aggregate nanomaterial.

[0031] The polypeptide A C -PFFRGD-NH2 is in dry powder form, A C After -PFFRGD-NH2 is dissolved in PBS solution or deionized water, the peptide concentration is 5 mg / mL.

[0032] The concentration of the PBS solution is 0.1-10 mM, and the pH value is 2-12.

[0033] The incubation temperature is 37° C., the incubation time is 1-6 days, and the rotation speed is 360 r / min.

[0034] The stable piezoelectric polypeptide aggregate nanomaterial formed by self-assembly has piezoelectric effect.

[0035] The method for using piezoelectric polypeptide aggregate nanomaterials for cell proliferation is carried out according to the following steps:

[0036] (1) Digest the cells in the growth phase during subculturing with trypsin, add double culture medium, transfer to a centrifuge tube, centrifuge at 1000 rpm / min for 5 min, and slowly pour off the supernatant; add 2-5 mL of culture medium, resuspend the cells, take 10-25 μL of cell culture medium, count with a cell counting plate, and dilute the cell concentration to 10,000 cells / mL as needed;

[0037] (2) Take 100 μL of the diluted cell suspension and add it to a 96-well plate. Add the cells to the 96-well plate and then place it in a 37°C carbon dioxide incubator for 24 h. Then replace the culture medium with A containing different concentrations. C -PFFRGD-NH2 nanomaterial culture solution, after shaking, continue to be placed in the incubator for 24-72h;

[0038] (3) After the culture in step (2) is completed, the culture medium in the above 96-well plate is replaced with a culture medium containing a cytotoxicity detection reagent CCK8; after CCK8 is completely reduced by dehydrogenase in the mitochondria to a highly water-soluble orange-yellow formazan product (Formazan), the absorbance value is measured using an enzyme reader under excitation at a wavelength of 450 nm.

[0039] The volume ratio of the water-soluble tetrazolium salt WST-8, the main chemical component of CCK8, and CCK8 to culture medium was 1:10;

[0040] At the same time, we took the absorbance value of the solution containing only culture medium and materials as the base value, excluding A C -The absorbance of PFRGD-NH2 nanomaterials themselves is affected.

[0041] In step (1), the cell is one of L929 and PC12.

[0042] Step (2) contains different concentrations of A C -PFFRGD-NH2 nanomaterial culture medium,

[0043] A C -PFFRGD-NH2 nanomaterial concentration is 0.025-0.125 mg / ml.

[0044] The beneficial effects of the present invention are:

[0045] The present invention synthesizes A by Fmoc solid phase chemical synthesis method C After the self-assembly process, the nanosheet-shaped aggregates formed by the -PFFRGD-NH2 hexapeptide molecules have a typical piezoelectric effect and have the function of adhering to cells and promoting cell proliferation. The present invention will provide potential application value for the development of biocompatible materials, as well as the fields of tissue engineering and regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A C -PFFRGD-NH2 hexapeptide molecule synthesized by Fmoc solid phase chemical synthesis method.

[0047] Figure 2 A C Transmission images of the nanostructured materials after incubation of -PFFRGD-NH2 polypeptide in PBS buffer with salt concentrations of 0 mM (A), 0.1 mM (B), 1 mM (C) and 10 mM (D) and pH 7 for 6 days.

[0048] Figure 3(A) hysteresis loop of nanosheet, (B) butterfly curve of nanosheet, (C) A C -Piezoelectric response curve of PFRGD-NH2 nanosheets.

[0049] Figure 4 A was incubated in PBS at pH 7, 10 mM for 1-6 days. C - Transmission image of PFRGD-NH2 polypeptide nanostructured material. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0051] Example 1

[0052] 1. A hexapeptide molecule A C -PFFRGD-NH2 preparation method, comprising the following steps:

[0053] (1) Resin pretreatment:

[0054] Weigh 1 g of 4-[(2,4-dimethoxyphenyl)(fluorenylmethoxycarbonyl-amino)methyl]phenoxyacetamidomethyl resin, put it into a vertical reactor, add dichloromethane DCM and soak for 30 minutes; then use a wash bottle to add a 20% piperidine / N,N-dimethylformamide (DMF) solution by volume to the reactor to completely soak the resin in the solution, react with nitrogen bubbling for 20 minutes, and then wash with DMF several times;

[0055] (2) The first amino acid is attached to the resin:

[0056] Weigh 0.3 mmol of aspartic acid derivative Fmoc-Asp(OTBU)-OH containing an amine protecting group Fmoc, add it to a centrifuge tube, dissolve it with DMF, then add 0.3 mmol N,N'-diisopropylcarbodiimide (DIC) and shake well, add it to the reactor in the previous step, react with nitrogen bubbling for 120 minutes, wash the resin with DMF several times after the reaction, add a mixed solution of acetic anhydride and DCM to the reactor, add 0.3 mmol N,N-diisopropylethylamine (DIEA) and react for 20 minutes.

[0057] (3) Washing

[0058] First, after the liquid in the reactor of step (2) is drained with a circulating water vacuum pump, DMF is added to the reactor with a wash bottle to completely soak the resin in the solution, and nitrogen bubbling is performed for 30 seconds. Then, the liquid in the reactor is drained with a circulating water vacuum pump for 30 seconds, and this is repeated 4 times;

[0059] Next, a 20% piperidine / DMF solution by volume was added to the reactor using a wash bottle to completely soak the resin in the solution, and nitrogen was bubbled for 20 minutes to remove Fmoc; after the reaction was completed, the liquid in the reactor was drained with a circulating water vacuum pump, and DMF was added to the reactor using a wash bottle to completely soak the resin in the solution, and nitrogen was bubbled for 30 seconds, and then the liquid in the reactor was drained with a circulating water vacuum pump, and the draining time was 30 seconds, and repeated 4 times;

[0060] Finally, the resin is used to detect whether the Fmoc removal is successful, and the resin is colored, indicating that the Fmoc removal is successful; and the resin A after being drained is obtained;

[0061] (4) The second amino acid is attached to the resin:

[0062] Weigh 1 mmol of Fmoc-protected glycine derivative Fmoc-Gly-OH and 1 mmol of 1-hydroxybenzotriazole HOBT into a centrifuge tube, fully dissolve them with DMF, add 1 mmol of DIC and mix, add to the reactor, mix with the dried resin A, and react for 1 hour with nitrogen bubbling;

[0063] Using resin detection to detect whether the amino acid is successfully connected, until the resin is colorless; washing the product using the washing method described in step (3), and draining to obtain resin B;

[0064] (5) The third, fourth, fifth, and sixth amino acids are attached to the resin:

[0065] Using the condensation method of step (4), the arginine derivative Fmoc-Arg(PBF)-OH containing the amine protecting group Fmoc, two phenylalanine derivatives Fmoc-Phe-OH, and the proline derivative Fmoc-Pro-OH are sequentially connected to the resin B, and after washing using the washing method described in step (3), the resin C is obtained after being dried;

[0066] (6) N-terminal acetylation modification of polypeptide chain:

[0067] A mixture of acetic anhydride and DCM was added to the reactor containing resin C to ensure that resin C was completely immersed, and 0.3 mmol DIEA was added to react for 20 min. After the reaction, the resin was washed several times with DMF and methanol, and then dried to obtain resin D.

[0068] (7) Cutting and purification:

[0069] Prepare a cutting reagent, including 95 ml trifluoroacetic acid (TFA) + 1 mL water + 2 mL 1,2-ethanedithiol (EDT) + 2 mL tris(hydroxymethyl)aminomethane (Tis), and precool anhydrous ether at -20°C for more than 2 hours;

[0070] Resin D was added to the cutting solution and cut for 2 h, then precipitated with ether;

[0071] The synthesized peptide was purified by high performance liquid chromatography (HPLC) to remove unreacted amino acids and other impurities; the purified peptide solution was freeze-dried to obtain the final peptide A. C -PFFRGD-NH2 powder.

[0072] The peptide on the resin was cut off and precipitated with ether and purified by high performance liquid chromatography, using 0.1% TFA-water solvent and 0.1% TFA-acetonitrile solvent as mobile phase A and mobile phase B, and collected and detected through the chromatographic column at a flow rate of 10 ml / min. The results are as follows Figure 1 The results show that the obtained A C The purity of -PFFRGD-NH2 polypeptide molecule is greater than 95%.

[0073] 2. A C -PFFRGD-NH2 hexapeptide self-assembly to form piezoelectric polypeptide aggregate nanomaterials:

[0074] A C -PFFRGD-NH2 hexapeptide powder was dissolved in a PBS solution with a salt concentration of 10 mM, the pH value of the PBS solution was adjusted to 7, and the solution was filtered through a sterile filter membrane before use to ensure that the peptide concentration in the solvent system was 5 mg / mL. Then, the solution was incubated in a constant temperature oscillator at 37°C and 360 r / min for 6 days to obtain a piezoelectric peptide aggregate nanomaterial, such as Figure 2 As shown in D.

[0075] The piezoelectric properties of the obtained nanomaterials were tested as follows: Figure 3 As shown in the figure. Since the hysteresis loop reflects the nonlinear hysteresis between the sample polarization and the applied electric field, the butterfly curve reflects the strain curve caused by the inverse piezoelectric effect and electrostrictive effect of the piezoelectric sample under the action of the applied electric field. Figure 3 From the results of A and B, both directly prove that A C -PFFRGD-NH2 nanosheets have piezoelectric properties. In addition, in the quantitative part, we obtained the curve of sample deformation versus voltage ( Figure 3 The slope is the piezoelectric coefficient d33 of the sample, which reflects the vertical strain caused by the electric field applied in the vertical direction. The piezoelectric coefficient of the polypeptide obtained in the experiment is d33 = 1.03 ± 0.06 pm V -1 In summary, the above experimental results show that A C -PFFRGD-NH2 nanosheets have stable piezoelectric properties.

[0076] 3. Application of piezoelectric polypeptide aggregate nanomaterials in cell proliferation:

[0077] (1) PC12 cells in the growth phase of subculturing were digested with trypsin and then added with double culture medium. The cells were transferred to a centrifuge tube and centrifuged (5 min, 1000 rpm / min). The supernatant was slowly poured off. 2 mL of culture medium was added to resuspend the cells. 10 μL of cell culture medium was taken and counted with a cell counting plate. The cell concentration was diluted to 10,000 cells / mL as needed.

[0078] (2) Take 100 μL of the diluted cell suspension and add it to a 96-well plate. Add the cells to the 96-well plate and then place it in a 37°C carbon dioxide incubator for 24 hours. Replace the culture medium and add A C -PFFRGD-NH2 nanomaterials, here directly 100 μL of pre-diluted nanosheet A containing 0.05 mg / ml C The cell culture medium of -PFFRGD-NH2 material was added to the 96-well plate. After shaking, the plate was placed in the incubator for 24 hours.

[0079] (3) After 24 hours, a 10% CCK8 solution was prepared and added to the 96-well plate in the form of a replacement solution for 4 hours. After all CCK8 was reduced by the dehydrogenase in the mitochondria to a highly water-soluble orange-yellow formazan product (Formazan), the absorbance value was measured using an ELISA reader under excitation at a wavelength of 450nm. At the same time, we used the absorbance value of the solution containing only the culture medium and the material as the base value to exclude the A C -The absorbance of PFRGD-NH2 nanomaterials themselves is affected.

[0080] Example 2

[0081] The same as Example 1, only the salt concentration of the PBS solution was changed to 0mM, 0.1mM, and 1mM. The shape of the obtained nanostructured material is as follows Figure 2 As shown in A, B, and C. From the results, it can be seen that in 0mM pure water solution, the short peptide is in the form of smaller oligomers; at a salt concentration of 0.1mM, some amorphous nano-aggregates appear; at a salt concentration of 1mM, the number of amorphous polypeptide aggregates increases and the size increases; finally, at a salt concentration of 10mM, A C -PFFRGD-NH2 short peptides assembled into well-structured two-dimensional multilayer nanosheets.

[0082] Example 3

[0083] The same as Example 1, only the incubation time was changed to 1, 2, 3, 4, 5 days, and the shape of the obtained nanostructured material was as follows Figure 4 As shown in the results, it can be seen that after one day of incubation, A C-PFFRGD-NH2 polypeptide also showed uniformly dispersed round nanoparticles under transmission electron microscopy, with a size of about 20nm. After 2 days of incubation, the nanoparticles were seen to aggregate to form fuzzy thin sheets with a certain thickness, and there were scattered nanoparticles distributed around the sheets. After 3 days of incubation, the thickness of the sheets aggregated by the nanoparticles increased, and the surrounding particles decreased, forming irregularly shaped nanoaggregates. On the 4th day, clearly visible nanosheets were found from TEM, and the size was about 500nm and had a certain thickness. On the fifth and sixth days of incubation, A C -PFFRGD-NH2 peptide will form thicker and larger nanosheets, which can reach about 2 microns. From the transparency of the edge part of TEM, it can be clearly seen that the nanosheets at this time are obviously multi-layered sheet structures.

[0084] Example 4

[0085] Same as Example 1, except that the A added in the cell proliferation experiment was changed. C The concentrations of -PFFRGD-NH2 nanomaterials were 0.025, 0.075, 0.1, and 0.125 mg / mL, and the resulting cell activity ratios are shown in Table 1.

[0086] Table 1 Different A C -Ratio of cell activity at different concentrations of PFRGD-NH2 nanomaterials

[0087]

Claims

1. A method for preparing a polypeptide molecule, characterized in that: The steps include: (1) Resin pretreatment: Weigh 4-[(2,4-dimethoxyphenyl)(fluorenylmethoxycarbonyl-amino)methyl]phenoxyacetamidomethyl resin, put it into a vertical reactor, add dichloromethane DCM and soak for 30-60 minutes; then use a wash bottle to add piperidine / N,N-dimethylformamide DMF solution to the reactor to make the resin completely soaked in the solution, react with nitrogen bubbling for 20-30 minutes, and then wash with DMF several times; (2) The first amino acid is attached to the resin: Dissolve the aspartic acid derivative Fmoc-Asp(OTBU)-OH containing the amine protecting group Fmoc in DMF, then add N,N'-diisopropylcarbodiimide DIC and shake well, add to the reactor of step (1), react with nitrogen bubbling for 120 to 150 minutes, wash the resin with DMF several times after the reaction, add a mixed solution of acetic anhydride and DCM to the reactor, and then add N,N'-diisopropylethylamine DIEA to react for 20 to 25 minutes; (3) Washing First, after the liquid in the reactor of step (2) is drained with a circulating water vacuum pump, DMF is added to the reactor with a washing bottle to completely immerse the resin in the solution, and nitrogen bubbling is performed for 30 to 40 seconds, and then the liquid in the reactor is drained with a circulating water vacuum pump for 30 to 40 seconds, which is repeated several times; Next, piperidine / N,N-dimethylformamide DMF solution was added to the reactor using a wash bottle to completely soak the resin in the solution, and nitrogen was bubbled for reaction for 20 to 25 minutes to remove Fmoc; after the reaction was completed, the liquid in the reactor was drained with a circulating water vacuum pump, and DMF was added to the reactor using a wash bottle to completely soak the resin in the solution, and nitrogen was bubbled for washing for 30 to 40 seconds, and then the liquid in the reactor was drained with a circulating water vacuum pump, and the draining time was 30 to 40 seconds, which was repeated several times; Finally, the resin is used to detect whether the Fmoc removal is successful, and the resin is colored, indicating that the Fmoc removal is successful; and the resin A after being drained is obtained; (4) The second amino acid is attached to the resin: The glycine derivative Fmoc-Gly-OH protected by the Fmoc group and 1-hydroxybenzotriazole HOBT were placed in a centrifuge tube in proportion and fully dissolved with DMF; N,N'-diisopropylcarbodiimide DIC was then added and mixed, and then added to the reactor, mixed with the resin A after being drained, and nitrogen bubbling was performed for condensation reaction for 1 to 1.5 hours; Using resin detection to detect whether the amino acid is successfully connected, until the resin is colorless; washing the product using the washing method described in step (3), and draining to obtain resin B; (5) The third, fourth, fifth, and sixth amino acids are sequentially connected to the resin: Using the condensation method of step (4), the arginine derivative Fmoc-Arg(PBF)-OH containing the amine protecting group Fmoc, two phenylalanine derivatives Fmoc-Phe-OH, and the proline derivative Fmoc-Pro-OH are sequentially connected to the resin B, and after washing using the washing method described in step (3), the resin C is obtained after being dried; (6) N-terminal acetylation modification of polypeptide chain: Add acetic anhydride and DCM mixture into the reactor containing resin C to ensure that resin C is completely immersed, add DIEA and react for 20 to 30 minutes. After the reaction, wash the resin several times with DMF and methanol, and then dry it to obtain resin D. (7) Cutting: Trifluoroacetic acid TFA, water, 1,2-ethanedithiol EDT and tris(hydroxymethyl)aminomethane Tis are used to prepare cutting reagents in proportion for use, and anhydrous ether is precooled at low temperature; Add resin D to the cleavage reagent and cleave for 2 to 2.5 hours, then precipitate with ether; The synthesized peptide was purified by high performance liquid chromatography (HPLC) to remove unreacted amino acids and other impurities; the purified peptide solution was freeze-dried to obtain the final peptide A. C -PFFRGD-NH2 powder.

2. The method for preparing a polypeptide molecule according to claim 1, characterized in that: In steps (1) and (2), the amount ratio of 4-[(2,4-dimethoxyphenyl)(fluorenylmethoxycarbonyl-amino)methyl]phenoxyacetamidomethyl resin, aspartic acid derivative Fmoc-Asp(OTBU)-OH containing amine protecting group Fmoc, N,N'-diisopropylcarbodiimide (DIC) and N,N-diisopropylethylamine (DIEA) is 1 g: 0.3 mmol: 0.3 mmol: 0.3 mmol.

3. The method for preparing a polypeptide molecule according to claim 1, characterized in that: In steps (1) and (4), the ratio of 4-[(2,4-dimethoxyphenyl)(fluorenylmethoxycarbonyl-amino)methyl]phenoxyacetamidomethyl resin, Fmoc-Gly-OH, 1-hydroxybenzotriazole HOBT and N,N'-diisopropylcarbodiimide DIC is 1 g: 1-1.5 mmol: 1 mmol: 1 mmol.

4. The method for preparing a polypeptide molecule according to claim 1, characterized in that: The ratio of 4-[(2,4-dimethoxyphenyl)(fluorenylmethoxycarbonyl-amino)methyl]phenoxyacetamidomethyl resin in step (1) and DIEA in step (6) is 1 g:0.3 mmol.

5. The method for preparing a polypeptide molecule according to claim 1, characterized in that: In steps (2) and (6), the volume ratio of acetic anhydride to DCM in the mixture of acetic anhydride and DCM is 1:5; In steps (1) and (3), the volume percentage of piperidine in the piperidine / N,N-dimethylformamide (DMF) solution is 20%; In step (7), in the cutting reagent, the volume ratio of trifluoroacetic acid TFA, water, 1,2-ethanedithiol EDT and tris(hydroxymethyl)aminomethane Tis is 95 mL:1 mL:2 mL:2 mL; the low temperature precooling temperature is -20°C, and the time is more than 2 hours.

6. A method for preparing a piezoelectric polypeptide aggregate nanomaterial, characterized in that: A polypeptide A prepared by the method according to any one of claims 1 to 5 C -PFFRGD-NH2 is self-assembled, and the specific steps are: Peptide A C -PFFRGD-NH2 is dissolved in a PBS solution or deionized water filtered through a sterile filter membrane, and then incubated in a constant temperature oscillator to obtain a piezoelectric polypeptide aggregate nanomaterial.

7. The preparation method according to claim 6, characterized in that: The polypeptide A C -PFFRGD-NH2 is in dry powder form, A C -PFFRGD-NH2 was dissolved in PBS solution or deionized water to a concentration of 5 mg / mL; The concentration of the PBS solution is 0.1-10 mM, and the pH value is 2-12; The incubation temperature is 37° C., the incubation time is 1-6 days, and the rotation speed is 360 r / min.

8. A piezoelectric polypeptide aggregate nanomaterial, characterized in that: It is prepared by the preparation method described in claim 6 and has piezoelectric effect.

9. The use of the piezoelectric polypeptide aggregate nanomaterial according to claim 8 for cell proliferation, characterized in that: The specific steps are: (1) Digest the cells in the growth phase during subculturing with trypsin, add double culture medium, transfer to a centrifuge tube, centrifuge at 1000 rpm / min for 5 min, and slowly pour off the supernatant; add 2-5 mL of culture medium, resuspend the cells, take 10-25 μL of cell culture medium, count with a cell counting plate, and dilute the cell concentration to 10,000 cells / mL as needed; (2) Take 100 μL of the diluted cell suspension and add it to a 96-well plate. Add the cells to the 96-well plate and then place it in a 37°C carbon dioxide incubator for 24 h. Then replace the culture medium with A containing different concentrations. C -PFFRGD-NH2 nanomaterial culture solution, after shaking, continue to be placed in the incubator for 24-72h; (3) After the culture in step (2) is completed, the culture medium in the above 96-well plate is replaced with a culture medium containing a cytotoxicity detection reagent CCK8; after CCK8 is completely reduced by the dehydrogenase in the mitochondria to a highly water-soluble orange-yellow formazan product, the absorbance value is measured using an enzyme reader under excitation at a wavelength of 450 nm.

10. The use according to claim 9, characterized in that The volume ratio of the water-soluble tetrazolium salt WST-8, the main chemical component of CCK8, and CCK8 to culture medium was 1:10; In step (1), the cell is one of L929 and PC12; Step (2) contains different concentrations of A C -PFFRGD-NH2 nanomaterial culture medium, A C -PFFRGD-NH2 nanomaterial concentration is 0.025-0.125 mg / ml.

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