Bioactive peptide and application thereof

Abstract

The invention discloses a bioactive peptide and application thereof, and belongs to the technical field of molecular biology, the bioactive peptide can deliver RNA fragments, FITC and adriamycin into cells in a transmembrane mode, and the bioactive peptide is safe and non-toxic to human cells and good in load delivery effect.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and specifically relates to a bioactive peptide with cell-penetrating effect. Background Art

[0002] The cell membrane is composed of a phospholipid bilayer, and its selective permeability makes it difficult for most exogenous substances (such as drugs, nucleic acids, fluorescent probes, etc.) to directly penetrate into the cell, severely limiting the research and application in the field of biomedicine. Traditional delivery technologies (such as liposome encapsulation, electroporation, etc.) often face problems such as low efficiency, large cell damage, or complex operation. Therefore, the development of efficient and safe transmembrane delivery tools has always been a research hotspot.

[0003] Cell-penetrating peptides (CPPs), as a class of short peptides that can carry target substances through the cell membrane, have attracted much attention due to their low immunogenicity and high compatibility. However, the existing transmembrane peptides still have significant defects: 1) Long natural sequences: such as the TAT peptide (11 amino acids) or Penetratin (16 amino acids), with high synthesis costs and insufficient structural stability; 2) Potential cytotoxicity: Some transmembrane peptides are prone to disrupting the cell membrane integrity due to their strong cationicity or hydrophobic aggregation properties, resulting in damage to normal cells; 3) Limited delivery efficiency: Most transmembrane peptides rely on energy-consuming endocytosis, and the target substances are easily trapped in endosomes / lysosomes, with a low actual cytoplasmic delivery rate; 4) Contradiction between loading and release: Although the covalent conjugation loading method can improve the delivery efficiency, it requires complex chemical modifications and is difficult to release the target substances; non-covalent binding is prone to delivery failure due to dissociation.

[0004] In addition, the existing transmembrane peptides have limited loading capacity for specific substances (such as RNA fragments, small molecule chemotherapeutic drugs), making it difficult to meet the needs of precision medicine. Therefore, there is an urgent need to develop a new type of transmembrane peptide with both high delivery efficiency, low toxicity, and simple structure to break through the technical bottleneck of transmembrane delivery. Summary of the Invention

[0005] To solve the deficiencies of the existing technology, the present invention proposes a bioactive peptide that is safe, non-toxic, can effectively load and mediate the delivery of target substances into cells.

[0006] The technical problems to be solved by the present invention are achieved through the following technical solutions: A bioactive peptide, whose amino acid sequence is as shown in SEQ IS No.1, from the N-terminus to the C-terminus is: Lys-Leu-Lys-Leu-Phe-Pro-Trp-Phe.

[0007] The bioactive peptide can bind to the target substance through covalent or non-covalent interactions, load and mediate the delivery of the target substance into cells.

[0008] In the present invention, the target substance is selected from the group consisting of RNA fragments, free fluorescein isothiocyanate (FITC), and doxorubicin.

[0009] In the present invention, the bioactive peptide is applied to the transmembrane cell delivery of target substances for non-disease diagnosis and treatment purposes.

[0010] In the present invention, the RNA fragment can be used to inhibit the proliferation, migration, and invasion of tumor cells. Doxorubicin is a cytotoxic agent used for anti-tumor treatment, and the bioactive peptide can be applied to the preparation of anti-tumor drugs.

[0011] In the present invention, the amino acid Lys-Leu-Lys-Leu-Phe-Pro-Trp-Phe (K-L-K-L-F-P-W-F) consists of positively charged residues (two lysines K contribute a +2 charge at physiological pH, making the peptide basic) and hydrophobic residues (leucine L, phenylalanine F, and tryptophan W can form a hydrophobic region). Proline W disrupts the regular secondary structure and introduces turns or bends, which enables the amphiphilicity brought about by the alternating arrangement of K and L to form hydrophilic and hydrophobic surfaces after forming an α-helix, showing the characteristics of a membrane-interacting peptide (cell-penetrating peptide). Therefore, it has the ability to penetrate cells and can be used for the transmembrane transport of drugs.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) Safe and non-toxic: The polypeptide has a molecular weight of about 1078 Da, consists of 8 amino acid residues, has no immunogenicity, and its residue sequence is derived from 20 natural essential amino acids, being safe and non-toxic to human cells. (2) Economical: The polypeptide can be prepared by the mature Fmoc solid-phase peptide synthesis method, with low cost and a mature quality control process, and the product is easily obtained. (3) Highly efficient and versatile: Compared with other cell-penetrating peptides, this polypeptide can efficiently enter cells in the form of covalent connection or covalent interaction loading of target substances, without the problem of difficult drug release.

[0013] Therefore, the present invention has a novel concept and ingenious design, and is a bioactive peptide that is safe and non-toxic, effectively loads, and mediates the delivery of target substances into cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the effect diagram of the polypeptide delivery labeled with FITC fluorescence of this application. DETAILED DESCRIPTION OF THE INVENTION

[0015] The following further describes the present invention in conjunction with specific embodiments, but does not limit the protection scope of the present invention thereby. Example

[0016] A bioactive peptide, whose synthesis process comprises the following steps: (1) Activating the resin: Weigh 3000 mg of Fmoc-Phe wang Resin, add 20 mL of methanol and soak for 60 min to fully swell it; (2) Solution replacement: After filtering the Fmoc-Phe wang Resin by suction, replace the methanol with acetonitrile, repeating three times; (3) Deprotection: Add 10 mL of acetonitrile solution containing 50% morpholine to the Fmoc-Phe wang Resin, blow it to boil by introducing carbon dioxide gas, maintain the reaction for 15 min, then filter it under pressure to remove the Fmoc group that has removed the amino group, wash the resin three times alternately with 10 mL of isobutanol and 10 mL of acetonitrile, and then use the ninhydrin method to detect that the resin should turn black or purple; (4) Condensation reaction: Connect the next amino acid. Weigh Fmoc-amino acid at 1.5 mmol / g resin, prepare 0.61 g of HOBs, 0.82 g of TBTU and 0.77 mL of DIPEA according to the HOBs / TBTU / DIPEA system, dissolve them in 20 mL of acetonitrile as the reaction solution, add the weighed Fmoc-amino acid, blow it to boil by introducing carbon dioxide gas at room temperature, maintain the reaction for 3 h. After the reaction, wash the resin three times alternately with 10 mL of isobutanol and 10 mL of acetonitrile, and detect the amino group; (5) Repeat steps (2)-(3), extend the polypeptide from the C-terminus to the N-terminus in the order of the polypeptide, repeat the processes of deprotection, washing, and condensation until the remaining amino acids are connected, and complete the connection of the polypeptide; (6) Solution replacement: After filtering the Fmoc-Phe wang Resin by suction, replace the acetonitrile with methanol, repeating three times; (7) Polypeptide cleavage: Dry the polypeptide-resin complex with carbon dioxide gas. Prepare 20 mL of a mixed cleavage reagent by mixing TFA / phenol / ultrapure water in a volume ratio of 90 / 8 / 2. Place the polypeptide-resin complex in a round-bottom flask, add the mixed cleavage reagent and stir magnetically for 3 h. Remove the resin with a 200-mesh sintered glass filter, drop the filtrate into frozen ethanol, and then centrifuge and precipitate at a speed of 5000 r / min and freeze-dry to constant weight to obtain the crude peptide; (8) Purify the crude peptide by high-performance liquid chromatography (HPLC) to make the purity of the polypeptide reach more than 95% to obtain the product.

[0017] The polypeptide product synthesized by solid phase according to the above method has an amino acid sequence (from the N-terminus to the C-terminus) of Lys-Leu-Lys-Leu-Phe-Pro-Trp-Phe. Example

[0018] The polypeptide product obtained in Example 1 was used for a cytotoxicity experiment (human non-small cell lung cancer cell A549). The protocol is as follows: (1) Inoculating cells: Take a 96-well plate and add the culture medium containing 1×10 4 human non-small cell lung cancer A549 cells to each well. Incubate in an incubator at 37 °C and 5% carbon dioxide for 24 h to allow the cells to adhere to the wall. (2) Culturing cells: Prepare a culture medium containing 0.5 mM of the polypeptide. Use the culture medium without the polypeptide as the control well (Control). Add them separately to the wells of the 96-well plate with inoculated cells to form a control, and then incubate in an incubator at 37 °C and 5% carbon dioxide for 48 h; (3) Color development: According to the MTT colorimetric method, add 20 μL of MTT to each well of the 96-well plate. After continuing to incubate for 3 h, discard the culture medium. Add 150 μL of DMSO to each well and shake for 10 min; (4) Colorimetry: Select a wavelength of 490 nm and calculate the average cell survival rate through the optical absorption value on an enzyme-linked immunosorbent assay detector. The results are shown in Table 1.

[0019] Table 1 Test results of polypeptide toxicity (human non-small cell lung cancer cell A549)

[0020] As can be seen from Table 1, the polypeptide obtained in Example 1 of the present invention was not found to cause obvious damage to human non-small cell lung cancer A549 cells under the conditions of high concentration (0.5 mM) and long time (48 h), and is safe and non-toxic. Example

[0021] The polypeptide product obtained in Example 1 was used for a cytotoxicity experiment (human fibroblasts). The protocol is as follows: (1) Inoculating cells: Take a 96-well plate and add the culture medium containing 1×10 4 human fibroblasts to each well. Incubate in an incubator at 37 °C and 5% carbon dioxide for 24 h to allow the cells to adhere to the wall. (2) Culturing cells: Prepare a culture medium containing 2 mM of the polypeptide. Use the culture medium without the polypeptide as the control well (Control). Add them separately to the wells of the 96-well plate with inoculated cells to form a control, and then incubate in an incubator at 37 °C and 5% carbon dioxide for 24 h; (3)Color development: According to the MTT colorimetric method, add 20 μL of MTT to each well of the 96-well plate, continue to incubate for 4 h, then discard the culture medium, add 150 μL of DMSO to each well, and shake for 10 min; (4)Colorimetry: Select a wavelength of 490 nm, and calculate the average cell survival rate through the light absorption value on an enzyme-linked immunosorbent assay (ELISA) immune detector. The results are shown in Table 1.

[0022] Table 2 Test results of polypeptide toxicity (human fibroblasts)

[0023] As can be seen from Table 2, the polypeptide obtained in Example 1 of the present invention was not found to cause obvious harm to human fibroblasts under the conditions of high concentration (2 mM) and long time (24 h), and was safe and non-toxic. Example

[0024] The polypeptide product obtained in Example 1 was used for the polypeptide delivery test (FITC), and the protocol was as follows: (1)Take a 96-well plate, add the culture medium of 1×10 4 human non-small cell lung cancer A549 cells to each well, and culture in an incubator at 37 °C and 5% carbon dioxide environment for 24 h to allow the cells to adhere to the wall; (2)Prepare a culture medium containing a certain concentration of polypeptide, add an appropriate amount of FITC for fluorescence labeling, and then incubate the human non-small cell lung cancer A549 cells with the FITC-labeled polypeptide culture medium. Use the FITC culture medium without polypeptide as a blank control. After incubating for 10 min, carefully aspirate the culture medium, and wash the adherent cells three times with PBS buffer; (3)Add a fixing solution to fix for 10 - 20 min. After fixing the cells, wash them 2 - 3 times with PBS buffer; (4)Add DAPI staining solution to stain for 5 - 10 min, and then wash it 2 - 3 times with PBS buffer; (5)Observe with a fluorescence microscope under the excitation lights of 488 nm and 543 nm respectively. The results are shown in Figure 1 .

[0025] From Figure 1 it can be seen that in only ten minutes, the FITC green fluorescence of human non-small cell lung cancer A549 cells is very obvious, indicating that the polypeptide has successfully and efficiently entered the cells. Example

[0026] The polypeptide product obtained in Example 1 was used for the polypeptide delivery test (FITC + RNA fragment), and the protocol was as follows: (1)Take a 96-well plate, add the culture medium of 1×10 4The culture medium of human non-small cell lung cancer A549 cells was cultured in an incubator at 37°C and 5% carbon dioxide for 24 h to allow the cells to adhere to the wall. (2) Human non-small cell lung cancer A549 cells were incubated with a medium containing 5 μg / mL FITC-labeled RNA fragment (UUUUUUUUUU) and 0.1 mM polypeptide. A medium containing only 5 μg / mL FITC-labeled RNA fragment was used as a control. After incubation for 30 min, the medium was carefully aspirated, and the adherent cells were washed three times with PBS buffer. (3) Fresh cell culture medium was added and cultured for 24 h. (4) 150 μL of DMSO was added to each well of a 96-well plate, shaken for 10 min, and centrifuged at 10000 r / min to obtain the supernatant, which was the intracellular extract. (5) HPLC was used to measure the concentration of the target substance in the intracellular extract. The specific test results are shown in Table 3.

[0027] Table 3 Test results of polypeptide-mediated RNA fragment delivery

[0028] As can be seen from Table 3, the polypeptide obtained in Example 1 of the present invention can efficiently mediate the delivery of RNA fragments into cells, and the intracellular RNA fragment concentration is increased by more than 18 times compared with the control group. Example

[0029] A polypeptide delivery test (adriamycin) was carried out using the polypeptide product obtained in Example 1. The protocol is as follows: (1) Take a 96-well plate and add 4 The culture medium of human non-small cell lung cancer A549 cells was cultured in an incubator at 37°C and 5% carbon dioxide for 24 h to allow the cells to adhere to the wall. (2) Human non-small cell lung cancer A549 cells were incubated with a medium containing 10 μg / mL adriamycin and 0.05 mM polypeptide. A medium containing only 10 μg / mL adriamycin was used as a control. After incubation for 60 min, the medium was carefully aspirated, and the adherent cells were washed three times with PBS buffer. (3) Fresh cell culture medium was added and cultured for 12 h. (4) 150 μL of DMSO was added to each well of a 96-well plate, shaken for 10 min, and centrifuged at 10000 r / min to obtain the supernatant, which was the intracellular extract. (5) HPLC was used to measure the concentration of the target substance in the intracellular extract. The specific test results are shown in Table 4.

[0030] Table 4 Test results of polypeptide-mediated adriamycin delivery

[0031] As can be seen from Table 4, the polypeptide obtained in Example 1 of the present invention can efficiently mediate the delivery of doxorubicin into cells, and the intracellular doxorubicin concentration is increased by more than 35 times compared with the control group.

[0032] In summary, the polypeptide Lys-Leu-Lys-Leu-Phe-Pro-Trp-Phe prepared by the Fmoc solid-phase peptide synthesis method shows no cytotoxicity to most cells including human non-small cell lung cancer A549 cells and human fibroblasts, is safe and non-toxic; and shows good transmembrane transport effects on RNA fragments, free fluorescein isothiocyanate (FITC), and doxorubicin.

Claims

1. A bioactive peptide, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1, from the N-terminus to the C-terminus: Lys-Leu-Lys-Leu-Phe-Pro-Trp-Phe.

2. The bioactive peptide according to claim 1, characterized in that: The bioactive peptide can load and mediate the delivery of a target substance into cells.

3. The bioactive peptide according to claim 2, wherein: The target substance is selected from the group consisting of RNA fragments, free fluorescein isothiocyanate, and doxorubicin.

4. Use of the bioactive peptide according to any one of claims 1-3 in transmembrane cell delivery of a target substance for non-diagnostic and non-therapeutic purposes.

5. Use of the bioactive peptide according to any one of claims 1-3 in the preparation of an anti-tumor drug.

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