A pH-responsive cell-penetrating peptide and its use

Abstract

The present invention discloses a pH-responsive cell-penetrating peptide and its uses. The cell-penetrating peptide can selectively cross cell membranes under pH control, exhibiting enhanced cell membrane-penetrating ability in an acidic environment (pH 6.2-6.4) compared to a neutral environment (pH 7.0-7.4). The cell-penetrating peptide can be used in an anti-tumor drug delivery system, enabling precise drug delivery in the acidic tumor microenvironment.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug delivery systems, and in particular relates to a pH-responsive cell-penetrating peptide and a use thereof. Background Art

[0002] Since the discovery that the first cell penetrating peptide (CPP), the transactivator of transcription (TAT), could cross the cell membrane, CPPs have garnered widespread attention as drug delivery vehicles. CPPs are typically short peptide sequences of 5-30 amino acids. Studies suggest that CPPs primarily deliver their bound substances into cells via endocytosis, direct penetration, or a variety of mechanisms. Compared with other drug delivery vehicles, CPPs offer advantages such as simple synthesis, excellent biocompatibility, minimal cell damage, post-cytosolic degradation, effective tissue penetration, and compatibility with various other drug delivery systems. Furthermore, they can be directly fused with bioactive proteins for recombinant expression, making them considered ideal delivery vehicles. Summary of the Invention

[0003] The object of the present invention is to provide a cell-penetrating peptide whose ability to cross cell membranes can respond to pH regulation and its use.

[0004] A pH-responsive cell-penetrating peptide, the amino acid sequence of which is shown in SEQ ID No: 1 in the sequence listing.

[0005] The pH-responsive cell-penetrating peptide comprises a hydrophobic end and a hydrophilic end, wherein the histidine component accounts for more than 40% of the total of the hydrophilic end and the hydrophobic end.

[0006] The pH-responsive cell-penetrating peptide has the ability to cross cell membranes within the pH range of 6.2-6.4.

[0007] The pH-responsive cell-penetrating peptide is used in the preparation of drugs for penetrating cell membranes within a pH range of 6.2-6.4.

[0008] The present invention has the following beneficial effects: The cell-penetrating peptides can selectively cross cell membranes under pH control, with enhanced cell membrane penetration in acidic environments (pH 6.2-6.4) compared to neutral environments (pH 7.0-7.4). These cell-penetrating peptides can be used in drug delivery systems to achieve precise drug delivery within the pH range of 6.2-6.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The figure is a comparison of the cell membrane-penetrating ability of the cell-penetrating peptides of the present invention at different pH values. DETAILED DESCRIPTION

[0010] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0011] Example 1: Screening and preparation of cell-penetrating peptides

[0012] The present invention designs a cell penetrating peptide containing both a hydrophobic end and a hydrophilic end, wherein the histidine component accounts for more than 40% of the total hydrophilic end and the hydrophobic end. The cell penetrating peptide is obtained by screening according to this principle.

[0013] The specific preparation method is as follows:

[0014] 1. Solvent treatment

[0015] DMF and methanol were soaked with G3 molecular sieves overnight before use to remove impurities and water.

[0016] 2. Full swelling of the resin

[0017] Weigh 2.0 g of amino resin into a clean, dry reaction tube, add 15 mL of DMF, and activate at room temperature for about 30 min.

[0018] 3. Connect the first amino acid

[0019] At room temperature, filter the solvent from the previous step through a sand core. Add 1 mmol of a 5-fold molar excess of the first C-terminal amino acid, a 5-fold molar excess of DMAP, and a 5-fold molar excess of DIC, using DMF as the solvent, and allow to react at room temperature for 3 hours. After the reaction is complete, wash the reaction 4-6 times with 5-6 mL of DMF each time. Then, add an appropriate amount of pyridine and acetic anhydride in a 1:1 volume ratio and allow to react for 30 minutes. After the reaction is complete, wash the reaction 4-6 times with 5-6 mL of DMF each time. (Purpose: To block any active spots on the unloaded resin.)

[0020] 4. Removal of Fmoc protecting group

[0021] Remove the solvent from the previous step by suction, add 10 mL of 20% piperidine DMF solution to the resin, stir under N2 for 10 minutes, filter out the solution, add 10 mL of 20% piperidine DMF solution, blow and stir under N2 for 5 minutes, and then filter out the solution. Repeat this operation twice, then wash with DMF 4 times and methanol 2 times, 5-6 mL each time.

[0022] 5. Ninhydrin detection removal effect

[0023] Take out a small amount of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN, and phenol solution, and heat at 105℃–110℃ for 5 minutes. If it turns dark blue, it is a positive reaction, indicating that the removal is complete and the next step can be carried out. If it is colorless, it means that the protecting group is not completely removed and the above deprotection operation needs to be repeated.

[0024] 6. Connect the second amino acid and remove the Fmoc protecting group

[0025] Weigh a 3-fold molar excess of the second amino acid at the C-terminus, a 3-fold molar excess of HBTU, and a 3-fold molar excess of HOBT into a reaction tube. Add an appropriate amount of DMF solution to completely dissolve it, then add a 10-fold molar excess of (pure) DIEA. React at room temperature for 40 minutes and wash with DMF 4-6 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin detection reagent. It turns colorless. Then add 10 mL of 20% piperidine DMF solution to remove Fmoc. Perform two times, 10 minutes and 5 minutes respectively. Then wash it with DMF 4 times and methanol 2 times, 5-6 mL each time. Take out a small amount of resin and test it with ninhydrin detection reagent. If it turns blue, it can proceed to the next step of the reaction.

[0026] 7. Repeat step 2.6 until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then drain.

[0027] 8. Add acetic anhydride in DMF solution and react for 2 hours, then drain.

[0028] 9. Resin shedding and pure product separation detection

[0029] Finally, the product was cut with trifluoroacetic acid cutting solution (95% TFA: 2% TIS: 2% EDT: 1% H2O) for 2 hours, and the reaction solution was filtered to obtain a trifluoroacetic acid solution of the polypeptide. The lysate was blown dry as much as possible with nitrogen, and then precipitated with ether, centrifuged, and then washed with ether 3 to 5 times to obtain a white solid. After dissolving in pure water, it was desalted and purified by HPLC, and the crystals were precipitated after freeze-drying, and a small amount was taken for MS analysis.

[0030] Example 2: Comparison of cell membrane penetration ability of cell-penetrating peptides at different pH values

[0031] 1. Coat slides, cover slips, six-well plates, etc. with 0.01 mg / ml PLL solution overnight.

[0032] 2. The next day, discard the PLL solution in the six-well plate and wash it three times with 75% alcohol for 3 minutes each time. Then blow dry it.

[0033] 3. Press 1×10 5 bEND.3 cells were seeded at a high density into six-well plates and the cells were observed to grow to about 70%-80% after 2-3 days.

[0034] 4. These pH-responsive CPPs were mixed with mouse nerve growth factor at a molar ratio of 20:1 (0.5 μg of mouse nerve growth factor), dissolved in 1 ml PBS at pH 7.4 and 6.3, placed in a 1.5 ml EP tube, and incubated at room temperature for 30 minutes.

[0035] 5. Discard the culture medium in the six-well plate, add 1 ml of the pre-formed complex solution into the six-well plate, and incubate in a 37% cell culture incubator for 30 minutes.

[0036] 6. Then add 1 ml of complete culture medium containing fetal bovine serum and place the cells in the incubator again for 4-6 hours.

[0037] 7. Remove the transfected cells, carefully aspirate the liquid from the six-well plate, add 2 ml of PBS buffer to each well, soak the coverslip for 5 minutes, and repeat 3 times.

[0038] 8. Aspirate the PBS used to remove the cells, add 2 ml of 4% paraformaldehyde to each well to soak the coverslip, and fix the cells in a 4°C refrigerator for 40 minutes.

[0039] 9. Aspirate 4% paraformaldehyde, add 2 ml of PBS to each well and soak the coverslip for 5 minutes. Repeat 3 times.

[0040] 10. Carefully remove PBS and add 0.3% Triton X-100 to each well to soak the coverslip for 15 minutes.

[0041] 11. Aspirate 0.3% Triton X-100, add 2 ml of PBS to each well and soak the coverslip for 5 minutes. Repeat three times to wash away excess Triton X-100 in the six-well plate.

[0042] 12. After removing PBS, add 1 ml of 5% blocking goat serum to each well to soak the coverslip and block at room temperature for 1 hour.

[0043] 13. Carefully aspirate the 5% blocking goat serum, place the coverslip on a glass slide with the cell side facing up, add 200 μL of the prepared primary antibody to each slide. The primary antibody is rabbit anti-mouse NGF monoclonal antibody (1:300), prepared with 1% blocking goat serum, place in a light-proof humid box, and incubate at 4°C overnight (incubation time 18h-20h).

[0044] 14. Carefully remove the primary antibody, place the coverslip back into the six-well plate, add 2 ml of PBS to each well and soak the coverslip for 5 minutes. Repeat 3 times to wash off excess primary antibody.

[0045] 15. Carefully remove the PBS and place the coverslip on a glass slide with the cell side facing up. Add 200 μL of the prepared fluorescent secondary antibody to each slide. The fluorescent secondary antibody is Alexa Fluor 488-labeled goat anti-rabbit fluorescent secondary antibody (1:500) prepared with 1% blocking goat serum. Place in a light-proof humid box and incubate in the dark at room temperature for 1 hour.

[0046] 16. Carefully aspirate the fluorescent secondary antibody, add 2 ml of PBS to each well and soak the coverslip for 5 minutes. Repeat 3 times to wash away excess fluorescent secondary antibody.

[0047] 17. Then add 500 μL of Hoechst 33342 nuclear dye (1:1000) to each well and incubate in the dark for 5 minutes.

[0048] 18. Carefully remove the nuclear dye, add 2 ml of deionized water to each well and rinse the coverslip for 5 minutes, rinse three times, carefully remove the excess deionized water with filter paper, drop 50 μL of mounting medium on a dry glass slide, place the coverslip on the mounting medium with the cells facing down to avoid bubbles, and seal the slide. After drying at room temperature away from light, apply nail polish on all sides to fix the coverslip.

[0049] 19. Observe using an inverted fluorescence microscope.

[0050] The results showed that ( Figure 1 ), cell-penetrating peptides can selectively cross the cell membrane under pH regulation, and have stronger cell membrane-crossing ability in an acidic environment of pH 6.3 than in a neutral environment of pH 7.4.

[0051] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A pH-responsive cell-penetrating peptide, characterized in that Its amino acid sequence is shown in SEQ ID No: 1 in the sequence listing.

2. Use of the pH-responsive cell-penetrating peptide according to claim 1 in the preparation of a drug carrier that can penetrate cell membranes within a pH range of 6.2-6.4.

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