Novel cell-penetrating peptide and uses thereof

Abstract

The present invention relates to a cell-penetrating peptide comprising an SR-rich amino acid sequence, a complex comprising the cell-penetrating peptide and a biologically active molecule bound thereto, and a composition for delivering a biologically active molecule comprising the complex. The cell-penetrating peptide according to the present invention has remarkably excellent cell permeability compared to conventional cell-penetrating peptides and does not exhibit cytotoxicity, and thus can be advantageously used in various fields such as diagnosis, drug delivery systems, recombinant protein vaccines, DNA / RNA therapeutic agents, and gene and protein therapy.

Description

Novel cell-permeable peptide and uses thereof

[0001] The present invention relates to a cell-permeable peptide comprising an SR-rich amino acid sequence, a complex comprising the cell-permeable peptide and a biologically active molecule bound thereto, and a composition for delivering a biologically active molecule comprising the complex.

[0002]

[0003] When a target substance for a disease is located inside a cell, intracellular delivery of the drug to that target substance is a prerequisite for the diagnosis, prevention, or treatment of the disease. Previously, research on intracellular drug delivery has been attempted using drug delivery systems such as electroporation, membrane fusion using liposomes, and nanoparticles. However, there were issues such as cytotoxicity, rapid loss by the body's immune system, and impairments caused by interactions with cells. To address these problems, many attempts have recently been made to utilize cell-penetrating peptides (CPPs).

[0004] Cell-permeable peptides are peptides composed of approximately 10 to 30 amino acids that have the function of transporting biologically active molecules, such as nucleic acids, proteins, and compounds, into cells without specific receptors. Although the amino acid sequences of each cell-permeable peptide vary, most contain a large number of basic amino acids (lysine and arginine), and some exhibit an amphipathic alpha-helical structure.

[0005] The most extensive research has been conducted on cell-permeable peptides derived from the TAT protein, a transcription factor of HIV-1 (human immunodeficiency virus-1); cell-permeable peptides derived from the homeodomain of the Antennapedia protein of Drosophila (penetratin); and the artificially designed cell-permeable peptide PEP-1. In addition, many other cell-permeable peptides have been reported and commercialized. However, it has been reported that cell-permeable peptides derived from viruses or Drosophila proteins may also induce an immune response, potentially leading to a reduction or loss of efficacy upon repeated administration. Furthermore, existing cell-permeable peptides have problems in that binding to biologically active molecules (cargo) alters the cell permeability and pattern of the peptide, resulting in the peptide failing to pass through the cell membrane or the activity of the biologically active molecule not being maintained. Moreover, the efficiency of penetrating the cell membrane and nuclear membrane was not actually high. In particular, considering that the efficiency of passing through the cell membrane decreases to less than half in environments where plasma or serum is present, there is a need to develop novel cell-permeable peptides that can efficiently pass through the cell membrane and be delivered into the cell even when bound to biologically active molecules.

[0006] Accordingly, the inventors completed the present invention by discovering that an SR-rich amino acid sequence can be used as a cell-permeable peptide due to effects such as cell permeability, ability to deliver biologically active molecules, and low cytotoxicity.

[0007]

[0008] The object of the present invention is to provide a cell-permeable peptide comprising an SR-rich amino acid sequence.

[0009] Another objective of the present invention is to provide a polynucleotide encoding the cell-permeable peptide.

[0010] Another objective of the present invention is to provide a complex comprising the cell-permeable peptide; and a biologically active molecule bound thereto.

[0011] Another objective of the present invention is to provide a composition for delivering biologically active molecules within a cell comprising the above complex.

[0012] Another objective of the present invention is to provide a method for delivering a biologically active molecule comprising the step of administering the complex to an individual or cell.

[0013]

[0014] To achieve the above objective, the present invention provides a cell-permeable peptide comprising an SR-rich amino acid sequence.

[0015] In addition, the present invention provides a polynucleotide encoding the cell-permeable peptide.

[0016] In addition, the present invention provides a complex comprising the cell-permeable peptide; and a biologically active molecule bound thereto.

[0017] In addition, the present invention provides a composition for delivering biologically active molecules within a cell comprising the above complex.

[0018] In addition, the present invention provides a method for delivering a biologically active molecule comprising the step of administering the complex to an individual or cell.

[0019]

[0020] The cell-permeable peptide according to the present invention has significantly superior cell permeability compared to conventional cell-permeable peptides and does not exhibit cytotoxicity, so it can be usefully used in various fields such as diagnosis, drug delivery systems, recombinant protein vaccines, DNA / RNA therapeutics, gene and protein therapies.

[0021]

[0022] Figure 1 shows the results of confirming the size and purity of GFP, GFP:SR12, and GFP:TAT using Coomassie staining.

[0023] Figure 2 shows the results of confirming the intracellular delivery efficiency of GFP according to the concentration of GFP:SR12 peptide (1 μM, 2 μM, and 5 μM) in U2OS cell lines (green: GFP; and blue: cell nucleus portion by DAPI staining).

[0024] Figure 3 shows the results of confirming cell permeability and intracellular delivery efficiency of GFP after treatment with 1 μM GFP, 1 μM GFP:SR12, and 1 μM GFP:TAT in U2OS cell lines (green: GFP; and blue: cell nucleus portion by DAPI staining).

[0025] Figure 4 shows the results of confirming cell permeability and intracellular delivery efficiency of GFP after treatment with 1 μM GFP, 1 μM GFP:SR12, and 1 μM GFP:TAT in HeLa cell lines (green: GFP; and blue: cell nucleus portion by DAPI staining).

[0026] Figure 5 shows the results of confirming cell permeability and intracellular delivery efficiency of GFP after treatment with 1 μM GFP, 1 μM GFP:SR12, and 1 μM GFP:TAT in WI-38 cell lines (green: GFP; and blue: cell nucleus portion by DAPI staining).

[0027] Figure 6 shows the results of confirming cell permeability and intracellular delivery efficiency of GFP after treatment with 1 μM GFP:SR6, 1 μM GFP:SR9, 1 μM GFP:SR12, and 1 μM GFP:SR20 in HeLa cell lines (Green: GFP; and Blue: cell nucleus portion by DAPI staining).

[0028]

[0029] The present invention will be described in detail below.

[0030] The terms used in this invention have been selected based on currently widely used general terms whenever possible, taking into account the functions of the invention; however, these terms may vary depending on the intent of those skilled in the art or the emergence of new technologies. Additionally, in specific cases, terms may be selected arbitrarily, and in such cases, their meanings will be described in detail in the description section of the relevant embodiments. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0031] In the present invention, when a component or step is described as "comprising," this means that, unless specifically stated otherwise, it does not exclude other components or steps but may include additional components or steps.

[0032]

[0033] The present invention provides a cell-permeable peptide comprising an SR-rich amino acid sequence.

[0034] The term "cell permeability" in the present invention refers to the ability or property of a substance to penetrate a cell (membrane) and infiltrate into the interior of the cell. The cell membrane refers to a lipid-containing barrier that separates a cell or a group of cells from the extracellular space. The cell membrane refers to a plasma membrane, a cell wall, an organelle membrane, such as a mitochondrial membrane or a nuclear membrane.

[0035] The term "peptide" in this invention refers to a polymer of amino acids; a form in which a small number of amino acids are linked is called a peptide, and a form in which many amino acids are linked is called a protein. In the structure of a peptide or protein, the connection between amino acids is formed by an amide bond or a peptide bond. A peptide bond refers to a bond in which water (H2O) is removed between a carboxyl group (-COOH) and an amino group (-NH2) to form a -CO-NH- structure.

[0036] The term "cell penetrating peptide (CPP)" of the present invention refers to a peptide having cell permeability, which has the ability to deliver a cargo into a cell in vitro and / or in vivo.

[0037] The above term, "cargo," includes all substances capable of moving into a cell by binding to a cell-permeable peptide, and may include all types of biologically active molecules without limitation. The said cargo includes, for example, all substances for which cell permeability efficiency is desired to be increased, specifically active substances of drugs, cosmetics, or health foods; more specifically, substances that are not easily moved into a cell through general routes; and even more specifically, proteins, nucleic acids, peptides, minerals, sugars such as glucose, nanoparticles, biological agents, viruses, contrast agents, or other chemical substances, but is not limited thereto, and may include without limitation any substances capable of moving into a cell and exerting biological activity.

[0038] In the present invention, the SR-rich amino acid sequence may be derived from an SR domain in which serine (S) and arginine (R) are repeated, present in SR splicing factors. Additionally, the SR-rich amino acid sequence may be synthesized by a gene synthesis method based on a nucleotide sequence or by other methods well known to those skilled in the art.

[0039] In the present invention, the SR-rich amino acid sequence may be (SR)n (n is an integer from 9 to 20). Preferably, the SR-rich amino acid sequence is SRSRSRSRSRSRSRSRSRSR (n=9), SRSRSRSRSRSRSRSRSRSRSR (n=10), SRSRSRRSRSRSRSRSRSRSSRSR (n=11), SRRSRSRSRSRSRSRSRSRSSRSRSR (n=12), SRSRSRRSRSRSRSRSRSRSRSSRSRSR (n=13), SRRSRSRSRSRSRSRSRSRSRSRSRSRSR (n=14), SRRSRSRSRSRSRSRSRSRSRSRSRSSRSRSR (n=15), SRRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSR (n=16), SRRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRS (n=17), SRRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRS (n=18), SRRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSSRSR (n=19) and It may include an amino acid sequence selected from a group consisting of SRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSRSR (n=20).

[0040] In the present invention, the cell-permeable peptide may mediate the transport of a biologically active molecule bound thereto into the cell.

[0041] In the present invention, the cell may be one or more selected from the group consisting of blood-brain barrier endothelial cells, cancer cells, blood cells, epithelial cells, skin cells, epidermal cells, dermal fibroblasts, cervical cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells, neural stem cells, T cells, B cells, natural killer cells, macrophages, monocytes, microglia, neurons, glial cells, astrocytes, muscle cells, brain cells, liver cells, kidney cells, lung cells, and laryngeal cells, but is not limited thereto.

[0042] In addition, the present invention provides a polynucleotide encoding the cell-permeable peptide.

[0043] In addition, the present invention provides a complex comprising the cell-permeable peptide and a biologically active molecule bound thereto.

[0044] In the present invention, the cell-permeable peptide may be bound to one end or both ends of a biologically active molecule.

[0045] In the present invention, the bond may be a chemical bond, a bond through a linker, or a peptide bond. The chemical bond may be one or more selected from the group consisting of disulfide bonds, diamine bonds, sulfide-amine bonds, carboxy-amine bonds, ester bonds, diselenide bonds, maleimide bonds, thioester bonds, and thioether bonds, but is not limited thereto.

[0046] In the present invention, the biologically active molecule may be one or more selected from the group consisting of peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, semi-synthetic drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, fluorochromes, and toxins, but is not limited thereto.

[0047] In addition, the protein may be one or more selected from the group consisting of growth factors, enzymes, nucleases, transcription factors, antigenic peptides, antibodies, antibody fragments, hormones, transport proteins, immunoglobulins, structural proteins, motor function proteins, receptors, signaling proteins, storage proteins, membrane proteins, transmembrane proteins, internal proteins, external proteins, secretory proteins, viral proteins, protein complexes, chemically modified proteins, and prions, but is not limited thereto.

[0048] In addition, the nucleic acid may be one or more selected from the group consisting of DNA, RNA, ASO (Antisense oligonucleotide), microRNA (microRNA, miRNA), small interfering RNA (siRNA), aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino, but is not limited thereto.

[0049] In addition, the above compounds may be one or more selected from the group consisting of therapeutic drugs and toxic compounds, but are not limited thereto.

[0050] In addition, the present invention provides a composition for delivering biologically active molecules within a cell comprising the above complex.

[0051] In addition, the present invention provides a method for delivering a biologically active molecule comprising the step of administering the complex to an individual or cell.

[0052] The above "individual" refers to a subject requiring treatment for a disease, and more specifically, to mammals such as human or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0053] The above "administration" means providing a specific composition or complex of the present invention to an individual by any appropriate method.

[0054] In the present invention, the complex may be administered at a concentration of 1 nM to 10 μM, but is not limited thereto.

[0055] In addition, the present invention provides a drug delivery composition comprising the cell-permeable peptide and a target drug.

[0056] In the present invention, the composition may be a composition for drug delivery into the skin, transdermally, or intracellularly.

[0057] The above drug may be an active ingredient or a physiologically active substance capable of exhibiting pharmacological activity that regulates activity within the skin, transdermis, or cells when delivered into the skin, transdermis, or cells. As an example, the above drug may be a compound, protein, nucleic acid, etc. As another example, it may include one or more selected from the group consisting of compounds such as charged polymeric compounds and fluorescent compounds; proteins such as enzymes, ligands, and antibodies; nucleic acids such as siRNA, plasmids, and genes; genetic materials; lipids, carbohydrates, dyes, photosensitizers, anticancer agents, antibiotics, chemical compounds, and combinations thereof, but is not limited thereto.

[0058] The above drug may be bound to or directly linked with a cell-permeable peptide through a linker, and the linker may be cleaved or degraded by various biological or chemical actions, such as enzymatic action, within the skin, transdermis, or cell, and depending on the cleaving or degradation of the linker, the peptide and the drug may be separated from each other within the target skin, transdermis, or cell.

[0059] The cell-permeable peptide of the present invention has the characteristic of being able to improve cell permeability without inhibiting the activity of the drug and having no cytotoxicity.

[0060]

[0061] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0062]

[0063] Example 1. Sequence synthesis of a cell-permeable peptide

[0064] Among the factors regulating RNA splicing, the SR splicing factor (SRSF) has an SR domain consisting of repeating serine and arginine. Accordingly, to determine whether the SR-rhch sequence could be used as a cell-permeable peptide, the inventors synthesized a peptide in which SR is repeated 12 times (hereinafter referred to as "SR12 peptide") based on the amino acid sequence of the SR domain of SRSF. The peptide was synthesized using a gene synthesis method based on the nucleotide sequence. In addition, a complex (hereinafter referred to as "GFP:SR12") was prepared by conjugating GFP (Green fluorescent protein), one of the biologically active molecules, to the SR12 peptide. GFP:SR12 was subjected to primary purification using bacteria, and secondary purification was performed using FPLC (Fast Protein Liquid Chromatography) to increase the purity of the peptide. After confirming the molecular weight of the peptides based on their amino acid sequences, they were stored in an ultra-low temperature freezer at -80°C until use. The size and purity of the purified peptides were confirmed using Coomassie staining. A GFP protein without the S12 peptide was used as the negative control, and a complex of TAT and GFP (hereinafter referred to as "GFP:TAT"), which is well known as a cell-permeable peptide, was used as the positive control.

[0065] As a result, as shown in Figure 1, GFP:SR12 was found to be approximately 31 kDa, which is slightly larger than the positive control GFP:TAT (29 kDa) and the negative control GFP (27 kDa). In addition, GFP:SR12 exhibited a high level of protein purity similar to GFP and GFP:TAT.

[0066]

[0067] Example 2. Confirmation of biologically active molecule delivery ability according to SR12 peptide concentration

[0068] To verify whether the SR12 peptide effectively delivers biologically active molecules into cells, experiments were conducted to confirm the translocation effect into cells using GFP fluorescent protein, a reporter protein, as an example of a biologically active molecule. GFP:SR12, in which GFP is conjugated to the SR12 peptide, was prepared at concentrations of 1 μM, 2 μM, and 5 μM, respectively, and human osteosarcoma cell lines, U2OS, were treated at each concentration. Briefly, 3 × 10⁶ U2OS cells were placed on a 4-well chamber slide. 4 Cells were seeded to a cell / well ratio and cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 for 20 hours to allow cell attachment to the chamber slide. Subsequently, GFP:SR12 was added at various concentrations and cultured for 1 hour. After the culture was terminated, the cells were washed three times with 1 mL of D-PBS to remove extracellular proteins. The washed cells were fixed using 4% paraformaldehyde and washed three more times with 1 mL of D-PBS. The nuclei of the fixed cells were stained using DAPI solution, mounted on VECTASHIELD® mounting medium, and analyzed using a confocal microscope.

[0069] As a result, as shown in Figure 2, it was clearly observed that fluorescence was emitted throughout the cell when treated with GFP:SR12, and it was confirmed that strong fluorescence was exhibited particularly in the nucleolus. This implies that GFP:SR12 can pass through the cell membrane, nuclear membrane, and even the nucleolus. Furthermore, it was confirmed that the fluorescence intensity increased as the treatment concentration of GFP:SR12 increased. This means that the number of GFP:SR12 molecules that can enter a unit cell increases in proportion to the concentration.

[0070]

[0071] Example 3. Evaluation of cell permeability efficiency of SR12 peptide using cell lines

[0072] 3.1. Evaluation of Cell Permeation Efficiency Using Human Osteosarcoma Cell Lines

[0073] To compare the cell permeability and delivery capacity of biologically active molecules of the synthesized SR12 peptide with TAT, a previously known cell-permeable peptide, GFP:SR12 (SR12 peptide conjugated with GFP) and GFP:TAT (TAT conjugated with GFP) were treated to the human osteosarcoma cell line U2OS, and the cell permeability efficiency was compared and analyzed. A GFP protein without SR12 was used as a negative control. Briefly, 3×10⁶ U2OS cells were placed on a 4-well chamber slide. 4Cells were seeded to a cell / well ratio and cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 for 20 hours to allow cell attachment to the chamber slide. Subsequently, 1 μM each of GFP, GFP:SR12, and GFP:TAT was added, and the cells were cultured for another 1 hour. After the culture was terminated, the cells were washed three times with 1 mL of D-PBS to remove extracellular proteins. The washed cells were fixed using 4% paraformaldehyde and washed three times again with 1 mL of D-PBS. The nuclei of the fixed cells were stained using DAPI solution, mounted on VECTASHIELD® mounting medium, and analyzed using a confocal microscope.

[0074] As a result, as shown in Figure 3, in the group treated with 1 μM GFP:SR12 for 1 hour, the cytoplasm, nucleoplasm, and nuclear membrane were clearly distinguishable, and fluorescence was observed throughout the cell. In contrast, it was confirmed that no fluorescence was observed in the cell with GFP. This implies that cell penetration is impossible with GFP alone, and that cell penetration occurred effectively through the SR12 peptide bound to GFP. Additionally, in the group treated with the positive control, GFP:TAT, faint fluorescence was observed throughout the cell without distinction between the nucleus and cytoplasm, and it was confirmed that the fluorescence intensity was very weak compared to GFP:SR12.

[0075] From the above results, it was confirmed that the SR peptide of the present invention has significantly higher cell permeability and biological molecule delivery efficiency into cells compared to the positive control TAT peptide in cancer cell lines such as osteosarcoma cell lines.

[0076]

[0077] 3.2. Evaluation of Cell Permeation Efficiency Using Human Cervical Cancer Cell Lines

[0078] In the same manner, the cell permeability and delivery capacity of biologically active molecules of the SR12 peptide were analyzed using the human cervical cancer cell line, HeLa. Briefly, 4×10⁶ HeLa cells were placed on a 4-well chamber slide. 4 Cells were seeded to a cell / well ratio and cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 for 20 hours to allow cell attachment to the chamber slide. Subsequently, 1 μM each of GFP, GFP:SR12, and GFP:TAT was added, and the cells were cultured for another 1 hour. After the culture was terminated, the cells were washed three times with 1 mL of D-PBS to remove extracellular proteins. The washed cells were fixed using 4% paraformaldehyde and washed three times again with 1 mL of D-PBS. The nuclei of the fixed cells were stained using DAPI solution, mounted on VECTASHIELD® mounting medium, and analyzed using a confocal microscope.

[0079] As a result, as shown in Figure 4, GFP:SR12, in which GFP is conjugated to the SR12 peptide, was observed to have distinct cytoplasm, nucleoplasm, and nuclear membrane, and to exhibit fluorescence throughout the cell.

[0080] From the above results, it was confirmed that the SR peptide of the present invention has significantly higher cell permeability and biological molecule delivery efficiency into cells compared to the positive control TAT peptide, even in cancer cell lines such as cervical cancer cell lines.

[0081]

[0082] 3.3. Evaluation of Cell Permeation Efficiency Using Human Fibroblast Cell Lines

[0083] In the same manner, the cell permeability and delivery capacity of biologically active molecules of the SR12 peptide were analyzed using the human fibroblast cell line WI-38. Briefly, WI-38 cells were placed on a 4-well chamber slide at a ratio of 3×10⁶ 4 Cells were seeded to a cell / well ratio and cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 for 20 hours to allow cell attachment to the chamber slide. Subsequently, 1 μM each of GFP, GFP:SR12, and GFP:TAT was added, and the cells were cultured for another 1 hour. After the culture was terminated, the cells were washed three times with 1 mL of D-PBS to remove extracellular proteins. The washed cells were fixed using 4% paraformaldehyde and washed three times again with 1 mL of D-PBS. The nuclei of the fixed cells were stained using DAPI solution, mounted on VECTASHIELD® mounting medium, and analyzed using a confocal microscope.

[0084] As a result, as shown in Figure 5, GFP:SR12, in which GFP is conjugated to the SR12 peptide, showed distinct separation of cytoplasm, nucleoplasm, and nuclear membrane, and fluorescence throughout the cell, similar to the results described earlier.

[0085] From the above results, it was confirmed that the SR peptide of the present invention has significantly higher cell permeability and biological molecule delivery efficiency into cells compared to the positive control TAT peptide, even in normal cell lines such as fibroblast cell lines.

[0086]

[0087] Example 4. Determination of the minimum number of SRs for the cell permeability of SR peptides

[0088] The SR12 peptide of the present invention is a peptide composed of a 24-amino acid sequence in which the SR sequence is repeated 12 times. Subsequently, the inventors further synthesized SR6, SR9, and SR20 peptides in which the SR sequence is repeated 6, 9, and 20 times, respectively, and conducted experiments to compare the cell permeability of a total of four types of peptides along with the SR12 peptide. The four types of SR peptides were bound to a GFP fluorescent protein to form a complex, and then experiments were conducted to confirm the migration effect into HeLa cells, a human cervical cancer cell line.

[0089] Briefly, 3×10⁶ WI-38 cells are placed on a 4-well chamber slide. 4 Cells were seeded to a cell / well ratio and cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 for 20 hours to allow cell attachment to the chamber slide. Subsequently, 1 μM of GFP:SR6, GFP:SR9, GFP:SR12, and GFP:SR20 were added, respectively, and cultured for another 1 hour. After the culture was terminated, the cells were washed three times with 1 mL of D-PBS to remove extracellular proteins. The washed cells were fixed using 4% paraformaldehyde and washed three times again with 1 mL of D-PBS. The nuclei of the fixed cells were stained using DAPI solution, mounted on VECTASHIELD® mounting medium, and analyzed using a confocal microscope.

[0090] As a result, as shown in Figure 6, cell penetration was observed for the SR9 peptide, which was repeated 9 times in addition to the SR12 peptide, when the microscope's Detector Gain value was increased to 800, and cell penetration was also observed for the SR20 peptide. However, it was confirmed that the delivery efficiency of the biologically active molecule was lower for the SR20 peptide due to its size.

[0091] From the above results, it was confirmed that the SR peptide of the present invention, a peptide in which the SR sequence is repeated 9 to 20 times, can be used as a cell-permeable peptide.

[0092]

[0093] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0094] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. Cell-permeable peptide containing an SR-rich amino acid sequence.

2. In Paragraph 1, The above SR-rich amino acid sequence is a cell-permeable peptide in which (SR)n (n is an integer from 9 to 20).

3. In Paragraph 1, The cell-permeable peptide described above is a cell-permeable peptide that mediates the transport of a biologically active molecule bound thereto into a cell.

4. In Paragraph 1, The cell-permeable peptide is one or more selected from the group consisting of blood-brain barrier endothelial cells, cancer cells, blood cells, epithelial cells, skin cells, epidermal cells, dermal fibroblasts, cervical cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells, neural stem cells, T cells, B cells, natural killer cells, macrophages, monocytes, microglia, neurons, glial cells, astrocytes, muscle cells, brain cells, liver cells, kidney cells, lung cells, and laryngeal cells.

5. A polynucleotide encoding a cell-permeable peptide according to any one of claims 1 to 4.

6. A complex comprising the cell-permeable peptide of claim 1; and a biologically active molecule bound thereto.

7. In Paragraph 6, A complex in which the cell-permeable peptide is bound to one or both ends of a biologically active molecule.

8. In Paragraph 7, A complex in which the above bond is a chemical bond, a bond through a linker, or a peptide bond.

9. In Paragraph 8, A complex in which the chemical bonds are one or more selected from the group consisting of disulfide bonds, diamine bonds, sulfide-amine bonds, carboxy-amine bonds, ester bonds, diselenide bonds, maleimide bonds, thioester bonds, and thioether bonds.

10. In Paragraph 6, A complex in which the above biologically active molecule is one or more selected from the group consisting of peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, semi-synthetic drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, fluorochromes, and toxins.

11. In Paragraph 10, The above protein is a complex comprising one or more selected from the group consisting of growth factors, enzymes, nucleases, transcription factors, antigenic peptides, antibodies, antibody fragments, hormones, transport proteins, immunoglobulins, structural proteins, motor function proteins, receptors, signaling proteins, storage proteins, membrane proteins, transmembrane proteins, internal proteins, external proteins, secretory proteins, viral proteins, protein complexes, chemically modified proteins, and prions.

12. In Paragraph 10, A complex in which the nucleic acid is one or more selected from the group consisting of DNA, RNA, ASO (Antisense oligonucleotide), microRNA (microRNA, miRNA), small interfering RNA (siRNA), aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino.

13. In Paragraph 10, A complex in which the above compound is one or more selected from the group consisting of therapeutic drugs and toxic compounds.

14. A composition for delivering biologically active molecules within a cell, comprising a complex according to any one of claims 6 to 13.

15. A method for delivering a biologically active molecule comprising the step of administering a complex according to any one of claims 6 to 13 to an individual or cell.

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