Cell-penetrating peptide and use thereof

Abstract

The application discloses a cell penetrating peptide and application thereof. The cell penetrating peptide is named cell penetrating peptide GAN_839K, and the amino acid sequence is RLWRIWHQNRRMRYRK. The cell penetrating peptide can penetrate the cell membrane of various types of cells to enter the cells, has the function of delivering proteins into the cells, and has no toxicity to the cells. The small peptide can be used for development of a drug delivery system and preparation of nanomaterials.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a cell-penetrating peptide and application thereof. BACKGROUND

[0002] Due to the barrier effect of cell membrane, a variety of proteins, polypeptides and oligonucleotides with therapeutic efficacy are difficult to enter cells to exert drug efficacy.

[0003] Cell-penetrating peptides (CPPs) are oligopeptides composed of about 8-30 amino acid residues, which can be absorbed by living cells. The protein, polypeptide and oligonucleotide drugs that are not easy to enter cells are coupled with cell-penetrating peptides, and then brought into cells by cell-penetrating peptides, so as to exert the function of protein, polypeptide and oligonucleotide drugs in cells.

[0004] There are different types of CPPs, including cationic peptides, amphipathic peptides (cationic / hydrophobic) and hydrophobic peptides (hydrophobic> cationic), in which cationic amino acids (such as arginine and lysine) play a key role.

[0005] For example, the application with publication number CN108707187A discloses a cell-penetrating peptide and a preparation method and application thereof, wherein the cell-penetrating peptide comprises a first sequence RRRRRKQARRPRRRRAR or a second sequence RSSRRRRRRRRRKQRKVKR.

[0006] For another example, the application with publication number CN108059655A discloses a cell-penetrating peptide and a preparation method and application thereof, wherein the cell-penetrating peptide comprises the following sequence: PGRKRRRRRRKG. The cell-penetrating peptide is safe and has a significant cell-penetrating effect.

[0007] Amphipathic peptide CPPs usually adopt a helical structure, and there is a clear separation between the cationic part and the hydrophobic part. The stable helical structure is the key to effective cell membrane permeability. CPPs can deliver non-membrane permeable compounds, proteins and nucleic acids into living cells; therefore, they are expected to be used as drug delivery tools. New CPPs with high efficiency and negligible toxicity are still under development (Oba M. Cell-Penetrating Peptide Foldamers: Drug-Delivery Tools. Chembiochem. 2019 Aug 16; 20(16): 2041-2045. doi: 10.1002 / cbic.201900204. Epub 2019 Jul 9. PMID: 30997711.). SUMMARY

[0008] The application discloses a novel cell-penetrating peptide which can efficiently enter various cells such as adherent cells, suspension cells and CD8 + T cells.

[0009] The application discloses a cell-penetrating peptide, which is named cell-penetrating peptide GAN_839K and has an amino acid sequence of RLWRIWHQNRRMRYRK.

[0010] Preferably, a maleimide modification is added to the C-terminal of the cell-penetrating peptide GAN_839K.

[0011] The application further provides application of the cell-penetrating peptide in preparation of a delivery system for delivering an exogenous protein into cells. + T cells.

[0012] The coupling between the polypeptide and the protein or other polypeptide can be in various modes.

[0013] Preferably, a maleimide modification is added to the C-terminal of the cell-penetrating peptide, and a cysteine is expressed at the C-terminal of the exogenous protein.

[0014] The cell-penetrating peptide disclosed by the application can penetrate the cell membrane and enter the cell, has the function of delivering the protein into the cell, and has no toxicity to the cell. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 GAN_839K polypeptide helical wheel structure schematic diagram, blue is the amino acid residue with a positive ion.

[0016] Figure 2A schematic diagram of the three-dimensional structure of GAN_839K polypeptide, in which the blue part is the N-terminal and the orange part is the C-terminal.

[0017] Figure 3 A schematic diagram of the coupling of GAN_839K polypeptide and EGFP protein by Michael addition method, in which "HA" represents that the C-terminal of EGFP is connected with an HA tag, "C" represents a cysteine residue, and "Mal" represents that a maleimide is modified at the C-terminal of CPP (here, GAN_839K polypeptide).

[0018] Figure 4 A plasmid map of EGFP protein.

[0019] Figure 5 A flow cytometry analysis result diagram for verifying the transfection experiment of GAN_839K polypeptide in adherent cells. The gray part is the flow cytometry result of GAN_839K polypeptide; the black part is the flow cytometry result of positive control (cationic peptide 9R), and **** represents p<0.0001.

[0020] Figure 6 A fluorescence result diagram for verifying the transfection experiment of GAN_839K polypeptide in adherent cells, in which A is the coupling product of GAN_839K polypeptide and EGFP; and B is EGFP without coupling GAN_839K polypeptide.

[0021] Figure 7 A flow cytometry analysis result diagram for verifying the transfection experiment of GAN_839K polypeptide in suspension cells. The gray part is the flow cytometry result of GAN_839K polypeptide; the black part is the flow cytometry result of positive control (cationic peptide 9R), and **** represents p<0.0001.

[0022] Figure 8 A fluorescence result diagram for verifying the transfection experiment of GAN_839K polypeptide in suspension cells, in which A is the coupling product of GAN_839K polypeptide and EGFP; and B is EGFP without coupling GAN_839K polypeptide.

[0023] Figure 9 A flow cytometry analysis result diagram for verifying the transfection experiment of GAN_839K polypeptide in CD8+ T cells. The gray part is the flow cytometry result of GAN_839K polypeptide; the black part is the flow cytometry result of positive control (cationic peptide 9R), and **** represents p<0.0001.

[0024] Figure 10 A fluorescence result diagram for verifying the transfection experiment of GAN_839K polypeptide in CD8+ T cells, in which A is the coupling product of GAN_839K polypeptide and EGFP; and B is EGFP without coupling GAN_839K polypeptide.

[0025] Figure 11 CCK-8 toxicity detection results of GAN_839K polypeptide in HEK 293T cells.

[0026] Figure 12 CCK-8 toxicity detection results of GAN_839K polypeptide in HeLa cells.

[0027] Figure 13 CCK-8 toxicity detection results of GAN_839K polypeptide in U87 cells.

[0028] Figure 14 CCK-8 toxicity detection results of GAN_839K polypeptide in HepG2 cells.

[0029] Figure 15 CCK-8 toxicity detection results of GAN_839K polypeptide in Jurkat cells.

[0030] Figure 16 CCK-8 toxicity detection results of GAN_839K polypeptide in K562 cells.

[0031] Figure 17 CCK-8 toxicity detection results of GAN_839K polypeptide in THP1 cells.

[0032] Figure 18 CCK-8 toxicity detection results of GAN_839K polypeptide in CD8+ T cells. DETAILED DESCRIPTION

[0033] Example 1: Coupling reaction of cell-penetrating peptide GAN_839K and EGFP protein

[0034] The present application first predicts the helical wheel structure (Fig. 1) and the three-dimensional structure (Fig. 2) of the cell-penetrating peptide GAN_839K polypeptide, Figure 1 Figure 2 determines that the polypeptide is an amphiphilic peptide and contains an α-helix secondary structure, which meets the physicochemical properties and structural characteristics of cell-penetrating peptides.

[0035] Figure 3 GAN_839K polypeptide and EGFP protein coupled by Michael addition method, wherein "HA" represents an HA tag connected to the C-terminal of EGFP (which can be used for subsequent detection of coupled products using antibodies against the HA tag), Figure 3 "C" on the right side of "HA" represents a cysteine residue, and "Mal" represents a maleimide modified at the C-terminal of the CPP (here, GAN_839K polypeptide). The EGFP amino acid sequence with an HA tag is shown in SEQ ID No. 1. ​

[0036] The present application adds a maleimide (Mal) modification ( Figure 3 ) to the C-terminal of the cell-penetrating peptide GAN_839K polypeptide (primary structure sequence RLWRIWHQNRRMRYRK), and expresses and purifies a green fluorescent protein (EGFP) containing a cysteine (C) at the C-terminal, Figure 4 The polypeptide (C-terminal modified with maleimide) synthesized by the company (Nanjing Jiepeikang Company) is coupled with the green fluorescent protein (C-terminal containing a cysteine) to deliver EGFP into cells.

[0037] 1. The original concentration of the cell-penetrating peptide GAN_839K is 4401.27 μM, and the concentration of the green fluorescent protein is 446 μM. The reaction system is 200 μL of green fluorescent protein slowly mixed with 121.60 μL of cell-penetrating peptide GAN_839K. The GAN_839K polypeptide and the EGFP protein are mixed at a molar ratio of 6:1. According to the molar ratio, the polypeptide is slowly added to the protein solution and mixed well. The reaction is carried out on an orbital shaker at room temperature for 2 hours.

[0038] 2. The sample after mixing and reaction is dialyzed at 4°C for 24 hours using a dialysis membrane with a 10kD cutoff.

[0039] 3. The concentration of the product at the end of dialysis is determined, and the filter membrane sterilization operation is carried out in a biological safety cabinet.

[0040] Example 2: Adherent cell delivery experiment

[0041] 1. HEK 293T cells, HeLa cells, U87 cells and HepG2 cells are cultured respectively, and the above four types of cells are cultured to a density of 70%-85%.

[0042] 2. Remove the culture medium of the above four types of cells, add DMEM complete medium mixed with GAN_839K-EGFP at a final concentration of 2 μM, and place the cells in a cell incubator at 37°C, 5% (volume concentration) carbon dioxide for 24 hours.

[0043] 3. Remove the polypeptide treatment solution, wash the cells with PBS twice, and then trypsinize and treat the cells. Resuspend the cells in DMEM complete medium for flow cytometry analysis.

[0044] 4. According to the method of Figure 5, GAN_839K polypeptide transfection experiment verification flow analysis results show that GAN_839K polypeptide than the positive control group polypeptide 9R (sequence is RRRRRRRRR, is the existing article reported can deliver Cas9 protein into the cell polypeptide. Article for: Suresh Ramakrishna, et al., (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA) protein delivery efficiency in HEK293T cells, HeLa cells, U87 cells and HepG2 cells, respectively, increased 1.5 times, 1.3 times, 2.2 times, 7.3 times, 2way ANOVA data analysis, p<0.0001, delivery efficiency is significantly improved.

[0045] 5、 Figure 6 GAN_839K polypeptide transfection experiment verification fluorescence results, wherein, A is GAN_839K polypeptide and EGFP coupling product; B is the EGFP of uncoupled GAN_839K polypeptide, from the figure, the GAN_839K polypeptide and EGFP coupling product treated cells can see green fluorescence, indicating that the EGFP protein has been successfully delivered into the cell; and the EGFP treated cells of uncoupled GAN_839K polypeptide can not see green fluorescence, indicating that the EGFP alone can not enter the cell.

[0046] According to Figure 5 、 Figure 6 The results prove that GAN_839K polypeptide can deliver proteins into adherent cells, and the protein delivery efficiency is increased by 1.3 to 7.3 times compared with the positive control group 9R, and the delivery efficiency is significantly improved.

[0047] Example 3: Suspension cell delivery experiment

[0048] 1. Cultivate Jurkat cells, K562 cells and THP1 cells respectively. On the day of cell treatment, the above three kinds of cells were inoculated at a cell density of 3x10 5 live cells / ml.

[0049] 2. After 2 hours of inoculation of the above three kinds of cells, 2 μM of GAN_839K-EGFP was added, and the cells were placed in a cell incubator at 37°C, 5% (volume concentration) carbon dioxide for 24 hours.

[0050] 3. Transfer the cells to a centrifuge tube and centrifuge at 800 rpm for 3 minutes to remove the peptide treatment solution. Wash the cells twice with PBS containing 2% FBS, and resuspend the cells in RPMI-1640 complete medium for flow cytometry analysis.

[0051] 4. According to Figure 7 Flow cytometry analysis of GAN_839K peptide demonstrated that its protein delivery efficiency was 12.3-fold, 6.9-fold, and 6-fold higher in Jurkat cells, K562 cells, and THP1 cells, respectively, compared to the positive control peptide 9R (sequence: RRRRRRRRR, a peptide previously reported to deliver Cas9 protein into cells, as described in the article "Suresh Ramakrishna, et al., (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA"). Two-way ANOVA analysis of the data revealed a significant improvement in delivery efficiency, with p < 0.0001.

[0052] 5. Figure 8 The fluorescence results of the membrane penetration experiment verification of GAN_839K polypeptide are shown, where A is the coupling product of GAN_839K polypeptide and EGFP; B is EGFP without GAN_839K polypeptide coupling. As can be seen from the figure, green fluorescence can be seen inside the cells treated with the coupling product of GAN_839K polypeptide and EGFP, indicating that the EGFP protein has been successfully delivered into the cells; while no green fluorescence can be seen inside the cells treated with EGFP without GAN_839K polypeptide coupling, indicating that EGFP alone cannot enter the cells.

[0053] according to Figure 7 、 Figure 8 The results showed that the GAN_839K polypeptide can deliver proteins into suspended cells, and the protein delivery efficiency is 6 to 12.3 times higher than that of the positive control group 9R, and the delivery efficiency is significantly improved.

[0054] Example 4: CD8 + T cell delivery experiments

[0055] 1. Cultivate CD8 + T cells. On the day of cell treatment, CD8+ T cells were cultured at a rate of 3×10 5 The cells were seeded at a density of 10 viable cells / ml.

[0056] 2. CD8 +Two hours after T cells were inoculated, GAN_839K-EGFP was added at a concentration of 2 μM, and the cells were placed in a cell culture incubator at 37° C. and 5% (volume concentration) carbon dioxide for 24 hours.

[0057] 3. Transfer the cells into a centrifuge tube and centrifuge at 1200 rpm for 3 min. Remove the peptide treatment solution, wash the cells twice with PBS containing 2% FBS, and resuspend the cells in AIM-V medium for flow cytometry analysis.

[0058] 4. According to Figure 9 The results of flow cytometry analysis of the membrane penetration test of GAN_839K polypeptide showed that the protein delivery efficiency of GAN_839K polypeptide was higher than that of the positive control polypeptide 9R (sequence: RRRRRRRRR, which is a polypeptide that can deliver Cas9 protein into cells as reported in the article: Suresh Ramakrishna, et al., (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA) in CD8 + The intracellular delivery of the drug increased by 8.2-fold, and the 2-way ANOVA data analysis showed a significant improvement in delivery efficiency with p < 0.0001.

[0059] 5. Figure 10 The fluorescence results of the membrane penetration experiment verification of GAN_839K polypeptide are shown, where A is the coupling product of GAN_839K polypeptide and EGFP; B is EGFP without GAN_839K polypeptide coupling. As can be seen from the figure, green fluorescence can be seen inside the cells treated with the coupling product of GAN_839K polypeptide and EGFP, indicating that the EGFP protein has been successfully delivered into the cells; while no green fluorescence can be seen inside the cells treated with EGFP without GAN_839K polypeptide coupling, indicating that EGFP alone cannot enter the cells.

[0060] according to Figure 9 、 Figure 10 The results showed that GAN_839K peptide can deliver proteins into CD8 + T cells, the protein delivery efficiency was 8.2 times higher than that of the positive control group 9R.

[0061] Example 5: Cytotoxicity Experiment

[0062] In this example, the cell viability was assessed using a CCK-8 assay to test whether GAN_839K was toxic to mammalian cells. CCK-8 is a commercially available test kit, and specific procedures are described in the relevant instructions.

[0063] Table 1

[0064] Polypeptide GAN_839K concentration (pM) 0.5 1 1.5 2 2.5 3 3.5 4 Toxicity on HEK 293T cells - - - - - - - - Toxicity on HeLa cells - - - - - - - - Toxicity on U87 cells - - - - - - - - Toxicity on HepG2 cells - - - - - - - - Toxicity on Jurkat cells - - - - - - - - Toxicity on K562 cells - - - - - - - - Toxicity on THP1 cells - - - - - - - - Toxicity on CD8+ T cells - - - - - - - -

[0065] Cytotoxicity results such as Figures 11-18 The results are summarized in Table 1. - indicates no toxicity, and + indicates toxicity. GAN_839K showed no toxicity to the above eight cell types within the concentration range tested.

Claims

1. A cell-penetrating peptide, characterized in that, designated cell-penetrating peptide GAN 839K, with the amino acid sequence RLWRIWHQNRRMRYRK.

2. The cell-penetrating peptide of claim 1, wherein, A maleimide modification is added to the C-terminal of cell-penetrating peptide GAN 474.

3. Use of the cell-penetrating peptide of claim 1 or 2 in the preparation of a delivery system for delivering an exogenous protein into a cell.

4. Use according to claim 3, characterized in that, The cell-penetrating peptide is coupled with the exogenous protein to be delivered.

5. Use according to claim 4, characterized in that, When coupling, a maleimide modification is added to the C-terminal of the cell-penetrating peptide, and a cysteine is expressed at the C-terminal of the exogenous protein, and the cell-penetrating peptide is coupled with the exogenous protein through Michael addition reaction of maleimide with thiol.

6. Use according to claim 5, characterized in that, When coupling, the molar ratio of the cell-penetrating peptide to the exogenous protein is 6:

1.

7. Use according to claim 5, characterized in that, When coupling, it is performed at room temperature.

8. Use according to claim 7, characterized in that, The coupling reaction time is not less than 2 hours.

9. Use according to claim 8, characterized in that, After coupling, the product is dialyzed to remove the cell-penetrating peptide that has not been successfully coupled.

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