Cell-penetrating peptides and their applications

Abstract

The present invention belongs to the field of biotechnology and relates to a cell-penetrating peptide and its application. The amino acid sequence of the cell-penetrating peptide is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 40. The polypeptide of the present invention is a functional active polypeptide based on food sources and has good human compatibility. It is derived from natural food proteins and human growth-related factors, is safe in source and non-toxic to cells, and solves the problem of difficult transmembrane absorption of current bioactive macromolecules. Therefore, it is a cell-penetrating peptide with a green source, good biocompatibility and high biosafety. It can be used in the development and application of pharmaceutical delivery systems, can be synthesized by chemical methods or biological methods, and the obtaining method is simple and efficient, greatly saving time and cost.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a cell-penetrating peptide and its application. Background Art

[0002] Biopharmaceuticals and drug carriers play an increasingly important role in the treatment of many diseases. However, cell membranes and most tissues have multiple barrier functions, making it difficult for larger molecules, as well as therapeutic polypeptides, proteins, oligonucleotides, and drugs, to enter the target site to exert their effects. At the same time, it is also difficult for various drug-loaded carriers to enter cells to release drugs. Only a small number of drug molecules enter diseased tissues and cells to play a therapeutic role. This natural defense mechanism can prevent the invasion of exogenous substances, but it also limits the therapeutic value of most available molecules and leads to an increase in the dosage and cost of drugs. Therefore, how to introduce bioactive molecules into cells and diseased sites is the focus of drug research. Especially in recent years, the research on drug target therapy has been extremely active. Due to its unique membrane transduction ability, cell-penetrating peptides can transfer various bioactive substances into cells and have also become the focus of target drug delivery research. Cell-penetrating peptides (CPPs) are oligopeptides composed of approximately 30 or fewer amino acid residues, have strong transmembrane ability, and can carry bioactive macromolecules with a molecular weight 100 times larger than their own into cells. CPPs can be conjugated with proteins, polypeptides, and oligonucleotide drugs that are not easily taken up by cells, and then bring bioactive molecules into cells, thereby exerting the functions of proteins, polypeptides, and oligonucleotide drugs inside cells (Nasrollahi, S.A., et al., Cell-penetrating peptides as a novel transdermal drug delivery system. Chem BiolDrug Des, 2012. 80(5): p. 639-46.; Gupta, B., T.S. Levchenko, and V.P.Torchilin, Intracellular delivery of large molecules and small particles bycell-penetrating proteins and peptides. Advanced Drug Delivery Reviews, 2005.57(4): p. 637-651.).

[0003] Currently, the methods used to screen cell-penetrating peptides are mainly display technologies, including phage display and mRNA display. Among them, phage display technology is more widely used in the screening and identification of CPPs. Phage display technology expresses exogenous polypeptides on the surface of phages to obtain specific artificial phages with linked genotypes and phenotypes, which then interact with target protein molecules or target cells on a solid phase.

[0004] The penetratin "RQIKIWFQNRRMKWKK" is one of the earliest discovered cell-penetrating peptides. It is a 16-mer peptide derived from the Drosophila antennapedia homeodomain. Penetratin is usually covalently linked to the surface of nanoparticles. One of the most common applications of penetratin-modified nanoparticles is to facilitate the delivery of drugs across the blood-brain barrier (Prochiantz, A. and A.A. DiNardo, Shuttling Homeoproteins and Their Biological Significance. Methods MolBiol, 2022. 2383: p. 33-44.). TAT is a transcriptional transactivator encoded by human immunodeficiency virus type 1 (HIV-1) that can effectively deliver proteins into cells at low concentrations. The TAT peptide "YGRKKRRQRRR" has been used to deliver β-galactosidase, horseradish peroxidase, ribonuclease, etc. into cells to study their functions and localizations in cells.

[0005] Cell-penetrating peptides can bind to a variety of biomolecules and have a very wide range of applications. They have been identified as safe and effective delivery vectors for delivering various cargos in vitro and in vivo, including peptides, proteins, small organic molecules, antisense oligonucleotides, fluorescent dyes, or siRNA. Among them, the most valuable is to promote the efficacy of drugs in disease treatment as a delivery vector (Kardani, K., et al., Cell penetrating peptides: the potent multi-cargo intracellular carriers. Expert Opinion on Drug Delivery, 2019. 16(11): p.1227-1258.; Liu, Y., Z. Zhao, and M. Li, Overcoming the cellular barriers and beyond: Recent progress on cell penetrating peptide modified nanomedicine in combating physiological and pathological barriers. Asian J Pharm Sci, 2022. 17(4): p. 523-543.). Related studies have shown that cell-penetrating peptides have great application potential in tumor treatment. The conjugation of doxorubicin with Penetratin can effectively induce apoptosis of tumor cell lines at a lower concentration (Lakkadwala, S., et al., Dual functionalized liposomes for efficient co-delivery of anti-cancer chemotherapeutics for the treatment of glioblastoma. J Control Release, 2019. 307: p. 247-260.).TAT-linked p53 protein (RI-TAT-p53C) and R11-linked p53 protein and hemagglutinin HA-2 (d11R-p53C-riHA2) can effectively promote apoptosis of tumor cells and prolong the lifespan of mice after intraperitoneal injection into a lymphoma mouse model (Snyder, E.L., et al., Treatment of Terminal Peritoneal Carcinomatosis by a Transducible p53-Activating Peptide. PLOS Biology, 2004. 2(2): p. e36.). Cell-penetrating peptides also serve as carriers for macromolecular nucleic acids such as oligonucleotides, including peptide nucleic acids (PNAs), siRNAs, plasmid DNAs, etc., and can target the cytoplasm or nucleus in the regulation of gene expression. The application of cell-penetrating peptides on nanocarriers cannot be underestimated. TAT linked to solid lipid nanoparticles is used for transdermal drug delivery. Compared with the control group, TAT-lipid nanoparticles increased the transdermal penetration ability of celecoxib by 3 to 6 times. Polymer micelles or nanoparticles modified with cell-penetrating peptides provide an effective means to improve the intracellular delivery of poorly soluble bioactive drugs and nucleic acid drugs. The conjugation of cell-penetrating peptides with siRNA combined with ultrasound nanoliposome technology is a novel siRNA delivery system that can effectively promote the clinical application of siRNA (Xie, X., et al., Efficient siRNA Delivery Using Novel Cell-Penetrating Peptide-siRNA Conjugate-Loaded Nanobubbles and Ultrasound. Ultrasound Med Biol, 2016. 42(6): p. 1362-74.).

[0006] The existing technologies mainly have the following disadvantages:

[0007] 1) The capacity of the polypeptide library of phage display technology is limited by the screening throughput. A large peptide library data will lead to difficult screening; the main reason is due to the limitation of transfection efficiency and it is extremely vulnerable to human factors, resulting in low screening efficiency.

[0008] 2) The screening cost of display technology is relatively high and there are many influencing factors, which easily lead to unstable results; the main reason is that display body technology mainly uses the specific binding of protein to protein or cell for screening, so the experimental cost is relatively high, and the stability of protein expression has a great impact on the results.

[0009] 3) Secondly, since these existing cell-penetrating peptides are derived from proteins expressed by viruses, other species (such as Drosophila), or are artificially synthesized based on the analysis of the amino acid sequences of previously known cell-penetrating peptides, the human safety of the transmembrane peptides is uncertain. When used in the human body, they may cause side effects such as immune responses. Summary of the Invention

[0010] First, based on existing machine learning and artificial intelligence tools, the present invention designs a cell-penetrating peptide library through an overlapping peptide library, and uses the MLCPP 2.0 and BChemRF-CPPred prediction tools to screen for cell-penetrating peptides that can be scored highly efficiently and quickly. Then, through cell experiments, cell-penetrating peptides with better permeability are further screened out. The screened cell-penetrating peptides can be used as drug delivery carriers and are widely used in the pharmaceutical field. To achieve the above object, the technical solution provided by the present invention is as follows:

[0011] The present invention first provides a cell-penetrating peptide, the amino acid sequence of which is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 40. Specifically, the amino acid sequence is shown as SEQ ID NO: 2-6, SEQ ID NO: 8-10, SEQ ID NO: 12, SEQ ID NO: 14-18, SEQ ID NO: 20-34 or SEQ ID NO: 36.

[0012] Preferably, the amino acid sequence is shown as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 38.

[0013] The present invention provides a coding polynucleotide of the cell-penetrating peptide described above.

[0014] The present invention further provides a delivery system for delivering substances such as drugs into cells, which contains the cell-penetrating peptide described above as a delivery carrier. Specifically, it is a liquid preparation or a dry powder preparation. Further, when administered, the concentration of the cell-penetrating peptide is 10 uM to 200 uM.

[0015] The present invention further provides the application of the cell-penetrating peptide or the cell delivery system described above in drug delivery.

[0016] Specifically, the drug is specifically a target protein, polypeptide, oligonucleotide, organic small molecule, antisense oligonucleotide, fluorescent dye, siRNA.

[0017] Preferably, the cell-penetrating peptide is conjugated with a target protein, polypeptide, and oligonucleotide drug, or directly mixed and administered; preferably, the cell-penetrating peptide is conjugated with a target protein to form a fusion protein, and is obtained by recombinant expression of its tandem-encoded nucleic acid; or obtained by chemical synthesis.

[0018] More specifically, when administered, the concentration of the cell-penetrating peptide in the preparation is 10 uM to 200 uM.

[0019] The present invention designs a cell-penetrating peptide library based on green-source proteins such as lactoferrin, human epidermal growth factor, and transforming growth factor, which greatly ensures the safety of the cell-penetrating peptide. The cell-penetrating peptide of the present invention can be used for the development of drug delivery systems and the preparation of nanomaterials, etc., and has good biocompatibility. Most of the existing cell-penetrating peptides currently come from viruses and other species, and are prone to immune responses when used in the human body, posing a greater safety hazard.

[0020] The cell-penetrating peptide of the present invention is a green cell-penetrating peptide with strong permeability and high safety. In particular, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 38 can penetrate the cell membrane and enter the cell, and its penetration effect is equivalent to or even 10 times higher than that of the Penetratin cell-penetrating peptide, and has no toxic or side effects on the cells.

[0021] The polypeptides and proteins or other polypeptides of the present invention can act directly by conjugation or simple mixing. There can be various different ways of conjugation. Since the cell-penetrating peptide is responsible for bringing the conjugate into the cell, and then the other half of the conjugate plays the intracellular function, so as long as the two can be conjugated together, the conjugation method is not particularly limited.

[0022] The polypeptide of the present invention is a functional active polypeptide with good human compatibility based on food sources, derived from natural food proteins and human growth-related factors, with a safe source and no toxicity to cells, solving the problem of difficult transmembrane absorption of current bioactive macromolecules. Therefore, a cell-penetrating peptide with a green source, good biocompatibility, and high biological safety is provided, and the target peptide can be obtained by chemical synthesis, biosynthesis, and purification and extraction, etc. The cell-penetrating peptide obtained by the present invention has a high cell uptake rate, can be used for the development and application of pharmaceutical delivery systems, can be synthesized by chemical or biological methods, and the obtaining method is simple and efficient, greatly saving time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a penetration effect diagram of CPP.

[0024] Figure 2For the penetration effect diagrams of different concentrations of CPP-31 and Pen.

[0025] Figure 3 It is the fluorescence result diagram for the verification of the cell-penetrating peptide transmembrane experiment. Specific implementation manners

[0026] The following further elaborates the present invention through specific embodiments, in order to better understand the present invention, but it does not constitute a limitation to the present invention.

[0027] Example 1: Peptide library establishment

[0028] 1) Determine the protein source

[0029] Lactotransferrin is a multifunctional globular protein that widely exists in various secretions of animals and humans. Lactotransferrin has various biological activities such as antibacterial, antioxidant, and anti-cancer. It is applied in products such as infant formula foods and cosmetics, and has high safety. It can be used as a source of human nutrition to supplement iron and amino acids, and can also be used as a drug for the prevention and treatment of various human diseases, for maintaining the balance of the intestinal flora, preventing infections and resisting viruses, inhibiting tumor occurrence and metastasis, and preventing free radicals generated in the body from damaging the body, etc.

[0030] Epidermal growth factor (EGF) is a heat-resistant single-chain low-molecular polypeptide. EGF has no glycosyl part, is very stable, heat-resistant and acid-resistant, and widely exists in the body fluids of animals and humans and in various glands. EGF can accelerate the generation of new cells, increase the number of new cells, promote the metabolism of skin cells, the dead skin layer gradually falls off, and keep the skin smooth. It has the effects of enhancing skin elasticity, reducing skin wrinkles and preventing skin aging. Therefore, EGF is often used to help tissue regeneration, repair body surface wounds and ulcers, and is applied in drugs and cosmetics.

[0031] Transforming growth factor β (TGF-β) belongs to the multifunctional cytokines of the transforming growth factor family and is a secreted protein. It can be regulated by immune cells for cell growth and differentiation, and its key role is to regulate the inflammatory process. It is currently mainly used as a cytokine to promote the differentiation of fibroblasts. Some studies also believe that TGF-β has potential application prospects in the treatment of wound healing, promoting cartilage and bone repair, and treating autoimmune diseases and transplant rejection through immunosuppression, etc.

[0032] The above-mentioned proteins are mostly distributed in the body fluids of humans and animals, and have a reparative effect. Moreover, lactotransferrin has been applied in foods. Based on the above-mentioned green and safe proteins derived from animals and humans, a cell-penetrating peptide library is designed.

[0033] 2) AI high-throughput screening of the peptide library

[0034] (1) Screen the above peptide library through the MLCPP 2.0 and BChemRF-CPPred servers. Set the polypeptide length range to 6 - 30 amino acids, and obtain the transmembrane strength and score of the above peptides through prediction and comparison.

[0035] (2) According to the CPP scoring threshold > 0.7 predicted in step (1), 40 peptide sequences were screened, and the results are shown in Table 1.

[0036] 3) Peptide synthesis

[0037] Synthesize the 40 cell-penetrating peptide sequences screened in step 1 by solid-phase chemical synthesis for subsequent experiments.

[0038] Table 1. Sequence information of 40 peptides

[0039] 。

[0040] Example 2 Screening and verification of cell-penetrating peptides based on cell models

[0041] Preparation of related reagents:

[0042] 1) Cells: Human keratinocytes (HaCaT)

[0043] 2) Preparation of cell-penetrating peptide stock solution: Use sterilized distilled water as the solvent to prepare a cell-penetrating peptide stock solution with a concentration of 3 mM screened in Example 1, and then aliquot and store it at -20 °C for later use.

[0044] 3) Cell lysis solution: 1M NaOH: Weigh 4.0 g of solid sodium hydroxide and make up to 100 mL of sodium hydroxide solution.

[0045] Experimental protocol:

[0046] Sample #1 Cytotoxicity detection

[0047] Seed HaCaT cells in a 96-well plate at a density of 1.0×10ˆ5. After culturing for 24 h until the cells are stable, add cell-penetrating peptides SEQ ID NO:1 - SEQ ID NO:40 at different concentrations (5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM) to the cells. Incubate at 37 °C and 5% CO2 for 24 h, then discard the culture medium. Add 90 μL of basal medium and 10 μL of CCK8 to each well, mix well, and incubate at 37 °C for 40 min. After incubation, measure the absorbance at 450 nm. Cell viability (%) = (OD of experimental group - OD of blank well) / (OD of blank control group - OD of blank well) * 100%. Cell viability < 70% indicates that the cell-penetrating peptide has cytotoxicity.

[0048] Experimental results:

[0049] The cytotoxicity test results of cell-penetrating peptides are shown in Table 2. The results indicate that cell-penetrating peptides (SEQ ID NO: 1 - SEQ ID NO: 40) are non-toxic to HaCaT cells within the tested concentration range.

[0050] Table 2 Cytotoxicity results of cell-penetrating peptides

[0051]

[0052] +: Cytotoxic; -: Non-cytotoxic.

[0053] Sample #2 Cell penetration assay

[0054] Cells were seeded in 12-well plates at a density of 1.5×10ˆ5. After 24 h of incubation until the cells were stable, the cells were washed twice with DMEM. Then, DMEM containing cell-penetrating peptides was added and incubated for 1 h. The final concentrations of CPP (SEQ ID NO: 1 - SEQ ID NO: 40) and Pen were 10 μM. After that, the cells were washed twice with PBS, and then 400 μL of cell lysis buffer (1 M NaOH) was added. After lysing at 4 °C for 10 min, the lysate was collected in an EP tube and centrifuged at 12000 rpm at 4 °C for 15 min. The supernatant was transferred to a new EP tube, and 100 μL was taken from each well. Three parallels were set for each group and transferred to a black 96-well microplate. The fluorescence intensity was read at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The protein concentration of the samples was determined by the Bradford method. The specific operation refers to the Bradford protein concentration assay kit. The relative fluorescence intensity was calculated based on the protein concentration of the samples, and the relative fluorescence intensity was expressed as the fluorescence intensity per milligram of protein.

[0055] Experimental results:

[0056] The results of detecting the penetration of cell-penetrating peptides by fluorescence spectrophotometry are as Figure 1 shown. At a polypeptide concentration of 10 μM, compared with the positive control polypeptide Pen, the penetration effects of CPP-3 (SEQ ID NO: 3), CPP-4 (SEQ ID NO: 4), CPP-16 (SEQ ID NO: 16), CPP-29 (SEQ ID NO: 29), and CPP-31 (SEQ ID NO: 31) are better than that of Pen (SEQ ID NO: 40), while the penetration effect of CPP-39 (SEQ ID NO: 38) is equivalent to that of Pen. Among them, the relative fluorescence intensity of CPP-16 (SEQ ID NO: 16) is 2.5 times that of Pen, and the relative fluorescence intensity of CPP-31 (SEQ ID NO: 31) is 4 times that of Pen, indicating that the penetration effect of CPP-31 (SEQ ID NO: 31) far exceeds that of Pen.

[0057] Cells were treated with CPP-31 (SEQ ID NO: 31) and Pen at different concentrations (10 μM, 100 μM, 200 μM) for 1 h, and then the relative fluorescence intensity was measured. The results are as Figure 2 shown. The penetration effects of CPP-31 and Pen were enhanced with the increase of concentration. Moreover, at the concentration of 100 μM, the relative fluorescence intensity of CPP-31 was nearly 10 times higher than that of Pen, indicating that CPP-31 could exhibit a penetration effect far exceeding that of Pen at low concentrations.

[0058] Example 3 Verification of the function of cell-penetrating peptide vector delivery

[0059] Preparation of related reagents:

[0060] 1) Cells: Human keratinocytes (HaCaT).

[0061] 2) Preparation of cell-penetrating peptide stock solution: Using sterilized distilled water as the solvent, a cell-penetrating peptide stock solution with a concentration of 3 mM was prepared, and then it was dispensed in small amounts and stored at -20 °C for later use.

[0062] 3) Hoechst33342: 1 mg of Hoechst33342 was dissolved in 1 mL of sterile water to prepare a 1 mg / mL mother liquor, and it was dispensed in small amounts and stored at -20 °C for later use.

[0063] 4) 4% Trypan blue: Weighed 0.4 g of trypan blue and dissolved it in 10 mL of sterile water to prepare a 4% trypan blue mother liquor, and it was dispensed in small amounts and stored at -20 °C for later use. Experimental protocol:

[0064] Sample #1 Conjugation reaction of cell-penetrating peptide and EGFP protein

[0065] Maleimide (Mal) modification was added to the C-terminus of the CPP polypeptide, and green fluorescent protein (EGFP) containing cysteine (C) at the C-terminus was expressed and purified. The two were conjugated. In this example, the Michael addition reaction of maleimide and thiol was used. CPP-31 was diluted to 1 M, and the concentration of green fluorescent protein was 446 μM. The reaction system was that 89.2 μL of cell-penetrating peptide CPP was slowly mixed into 200 μL of green fluorescent protein. The CPP-31 polypeptide and EGFP protein were mixed at a molar ratio of 6:1. According to the molar ratio, the polypeptide was slowly added to the protein solution and mixed evenly, and the reaction was carried out on a shaker at room temperature for 2 h. Then, the mixed reaction sample was dialyzed at 4 °C for 24 h using a dialysis membrane with a cut-off molecular weight of 10 kD. The concentration of the product was measured, and after filtering the bacteria with a filter membrane, it was stored at -80 °C.

[0066] Sample #2 Delivery experiment

[0067] Calculate the concentration of the sample after sterilization, and dilute CPP-EGFP to a final concentration of 10 μM. Seed the cells in a 12-well plate at a density of 1.5×10ˆ5. After culturing for 24 h until the cells are stable, wash the cells twice with DMEM, and then add DMEM containing cell-penetrating peptide and incubate for 1 h. Add 10 μL of Hoechst 33342 to the wells and stain for 10 min, with a final concentration of 10 μg / mL. Remove the solution, add 300 μL of 150 μM trypan blue solution to treat the cells for 30 s, wash twice with PBS, and add 500 μL of medium to observe the imaging under a fluorescence microscope.

[0068] Experimental results:

[0069] As Figure 3 shown, where A is the conjugate of CPP-31 and EGFP; B is EGFP without conjugated CPP-31 polypeptide. It can be seen from the figure that obvious green fluorescence can be observed in the cells treated with the conjugate of CPP-31 polypeptide and EGFP, indicating that EGFP is delivered into the cells. However, no green fluorescence can be seen inside the cells treated with EGFP without conjugated CPP-31 polypeptide, indicating that free EGFP does not enter the cells.

Claims

Use of a cell-penetrating peptide having an amino acid sequence as shown in SEQ ID NO. 31 as a delivery system carrier in the preparation of a formulation of a drug delivery system.

2. The application according to claim 1, wherein The drug is a target protein, polypeptide, oligonucleotide, organic small molecule, antisense oligonucleotide, fluorescent dye or siRNA.

3. The application according to claim 2, wherein, The cell-penetrating peptide is conjugated with a target protein, polypeptide and oligonucleotide drug, or directly mixed and administered.

4. The application according to claim 2, wherein The cell-penetrating peptide is conjugated with a drug, or recombinantly expressed through its tandem encoding nucleic acid.

5. The application according to claim 4, characterized in that The conjugation method is amine conjugation of an amino acid residue of lysine, thiol conjugation of a cysteine residue, carbodiimide method, maleimide method, glutaraldehyde method.

6. The application according to claim 5, wherein When administered, the concentration of the cell-penetrating peptide in the formulation is 10 uM to 200 uM.

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