New cell penetrating peptides and uses thereof

Abstract

The present application relates to a kind of new cell penetrating peptides and its use. It is proved that the 12 cell penetrating peptides of the present application have excellent cell penetration ability and mass transfer effect in vitro and in vivo, since the above-mentioned cell penetrating peptides can effectively deliver biologically active substances to cells, tissues and other in vivo, and are expected to be widely used in research fields, various disease diagnosis and treatment fields, etc.

Description

TECHNICAL FIELD

[0001] The present application relates to an intracellular delivery technology for delivering a bioactive substance into a cell, and more particularly, to a novel cell-penetrating peptide having excellent cell-penetrating ability and use thereof. BACKGROUND

[0002] So far, researches on intracellular delivery of various low molecular compounds, high molecular substances such as proteins, peptides, RNA, DNA, etc. and their applications have been continuously conducted, and in particular, attempts have been made to regulate intracellular functions by delivering various proteins such as SOD, Catalase, SOCS, etc. inhibitory form proteins of intracellular signal transduction proteins such as dnPI3K, ZAP70 mutant, transcription factor proteins or DNA binding domain portions of transcription factors such as Foxp3, RORgt, etc. In addition, attempts have been made to deliver low molecular compounds such as cyclosporin A, etc. into cells and tissues to regulate rejection reactions occurring when transplanting organs and cells and to regulate autoimmune diseases such as psoriasis.

[0003] However, substances generally having hydrophilicity or a large molecular weight cannot enter into a cell due to a barrier called a cell membrane. The cell membrane prevents macromolecules such as peptides or proteins, nucleic acids, etc. from entering into a cell, and even if they enter into a cell, they are fused with lysosomal compartments of a cell and finally decomposed due to a physiological mechanism of endocytosis of a cell membrane receptor, and thus there are many limitations in disease treatment and prevention using the above-described macromolecules. In addition, in the case of anticancer drugs, for example, it is necessary to overcome problems such as multidrug resistance in order to deliver the drugs into cells. Thus, in order to prevent decomposition of the drugs, various methods of directly delivering carriers containing various macromolecules and drugs into a cell while bypassing endocytosis have been proposed. The above-described methods include microinjection, electroporation, etc., but these methods can damage the cell membrane. Another method includes a method of using a cell-penetrating substance, etc. However, even if the drugs are delivered into a cell by the above-described methods, there is a problem that the drugs must be transferred to a specific organ in order to exert a pharmaceutical effect.

[0004] Accordingly, various drug carriers capable of overcoming the above problems and improving stability and mass transfer efficiency have been developed, and representative examples include liposomes and micelles. Liposomes are artificially manufactured phospholipid carriers, which can encapsulate lipophilic and hydrophilic drugs and are biocompatible substances, and thus are non-toxic and can protect drugs from the external environment. However, they have disadvantages of delayed absorption, limited distribution, reduced metabolic rate, and rapid removal from the blood by being captured by liver or spleen cells. Micelles have characteristics capable of improving drug solubility and in vivo utilization, but many studies on the transfer effect of substances into cells and basic medical and clinical applicability are still required. Due to the above limitations, new formulations are required, which can effectively deliver biomaterials into the body without cytotoxicity, in particular, without entering the body through endocytosis.

[0005] Accordingly, cell penetrating peptides have attracted attention as a new alternative. Cell penetrating peptides are a kind of signal peptides, which are peptides composed of specific amino acid sequences, and are used to deliver macromolecular substances such as proteins, DNA, RNA, etc. into cells. For example, it was found in the 1990s that an 11-amino acid sequence present in the TAT protein derived from the HIV virus can deliver β-galactosidase into cells and tissues, and since then, related research has officially begun. Antennapedia (Penetratin) derived from a fruit fly protein, VP22 derived from the HSV-1 virus (Elliott, G. et al., Cell, 88:223, 1997), and Pep-1 derived from the SV40 large T antigen are considered to be typical first-generation cell penetrating peptides, and are widely used together with TAT. In addition, it has been reported that peptides in which a plurality of cationic amino acids such as arginine and lysine are repeatedly linked (e.g., polyarginine, polylysine, etc.) also have excellent cell penetration ability, and are used for various mass transfer. However, these cell penetrating peptides are mostly considered to have the potential for immunogenicity and toxicity, and have poor efficacy in delivering to human cells. Accordingly, there is a need to develop a cell penetrating peptide that does not cause toxicity, has in vivo safety, and can effectively transfer mass. SUMMARY

[0006] TECHNICAL PROBLEM

[0007] The present inventors have made strenuous efforts to develop a new cell penetrating peptide capable of overcoming the above problems, and finally designed and synthesized a new peptide having superior cell penetration ability to existing cell penetrating peptides, and confirmed that the 2nd and 6th amino acids of the peptide are important amino acids determining cell penetration ability through in vitro and in vivo experiments, thereby completing the present application based on the same.

[0008] Accordingly, an object of the present application is to provide a novel cell penetrating peptide.

[0009] In addition, another object of the present application is to provide a complex including the cell penetrating peptide and a bioactive substance.

[0010] In addition, another object of the present application is to provide a composition for mass transfer including the complex and a method for mass transfer including treating cells with the composition.

[0011] However, the technical problems to be solved by the present application are not limited to the above-mentioned problems, and other technical problems not mentioned will be clearly understood by those skilled in the art through the following description.

[0012] Technical Solution

[0013] In order to achieve the objects of the present application as described above, the present application provides a cell penetrating peptide consisting of an amino acid represented by [Formula I].

[0014]

Formula I

[0015] X1-X2-...X n-1 -X n

[0016] In the Formula I,

[0017] n≥16,

[0018] X2 is His (H) and X6 is Leu,

[0019] The amino acid other than X2 and X6 can be any one selected from the group consisting of glycine (Gly, G), histidine (His, H), glutamic acid (Glu, E), arginine (Arg, R), lysine (Lys, K), serine (Ser, S), aspartic acid (Asp, D), tryptophan (Trp, W), valine (Val, V), threonine (Thr, T), alanine (Ala, A), asparagine (Asn, N), and tyrosine (Tyr, Y).

[0020] As an embodiment of the present application, n in the Formula I can be 16.

[0021] As another embodiment of the present application, the cell penetrating peptide can consist of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

[0022] As another embodiment of the present application, the cell can be selected from the group consisting of a brain endothelial cell, a cancer cell, a blood cell, a lymphocyte, an immune cell, a stem cell, an induced pluripotent stem cell (iPSC), a neural stem cell (NSC), a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a microglia, a neuron, an astrocyte, and a muscle cell.

[0023] In addition, the present application provides a complex including the cell-penetrating peptide and a bioactive substance.

[0024] As an embodiment of the present application, the bioactive substance can be at least one selected from the group consisting of a compound, a protein, a glycoprotein, a peptide, an antibody, an enzyme, a nuclease, a hormone, a cytokine, a transcription factor, a toxin, a nucleic acid, a carbohydrate, a lipid, a glycolipid, a natural product, a semisynthetic drug, a drug, a microparticle, a nanoparticle, a liposome, a virus, a quantum dot, and a fluorescent dye.

[0025] As another embodiment of the present application, the nuclease can be selected from the group consisting of CAS9, CAS12, CAS13, CAS14, CAS variants, Cfp1 (CxxC-finger protein-1), ZEN (Zinc-finger nucleases), and TALEN (Transcription activator-like effector nuclease).

[0026] As another embodiment of the present application, the nucleic acid can be selected from the group consisting of DNA, RNA, an antisense oligonucleotide (ASO), a microRNA (miRNA), a small interfering RNA (siRNA), a nucleic acid aptamer, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), and a morpholino.

[0027] In addition, the present application provides a composition for mass transfer including the complex.

[0028] In addition, the present application provides a mass transfer method including treating a cell with the composition.

[0029] Advantageous effects

[0030] The present application synthesizes a new cell penetrating peptide, which has excellent cell penetration ability and mass transfer effect in vivo and in vitro, which is confirmed by experiments. The cell penetrating peptide according to the present application can effectively deliver a substance having a biological activity to a cell, a tissue, etc. in vivo, and thus is expected to be widely used in research fields, diagnosis and treatment fields of various diseases, etc. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a result of verifying whether it has in vitro cell penetration by treating cells with a substance synthesized by connecting 12 peptides with fluorescein isothiocyanate (FITC) at a concentration of 4 uM.

[0032] FIG. 2a is a result of analyzing in vitro cell penetration of a variant peptide in which a single mutation is induced to the amino acid of the 2nd and 6th positions according to the present application, as shown in Table 2, in order to analyze the effect of the amino acid of the 2nd and 6th positions of the cell penetrating peptide according to the present application on cell penetration.

[0033] FIG. 2b is a result of analyzing in vitro cell penetration of a variant peptide in which double mutations are induced to the amino acid including the above two positions, as shown in Table 3, in order to analyze the effect of the amino acid of the 2nd and 6th positions of the cell penetrating peptide according to the present application on cell penetration.

[0034] FIG. 3a is a result of analyzing in vivo fluorescence images of the penetration ability and mass transfer effect on the cerebral cortex and hippocampus tissues of a mouse after preparing a peptide fusion by binding GFP to the peptide of SEQ ID NO: 1, which is representative of the cell penetrating peptide according to the present application.

[0035] FIG. 3b shows a quantification result of the fluorescence images in the above-described FIG. 3a.

[0036] Figure 4 is a result of comparing and analyzing the cell penetration of angiopep-2, which is a cell penetrating peptide known in the art, and transpap-1 according to the present application by flow cytometry using transpap-1-FITC and angiopep-2-FITC.

[0037] FIG. 5a is a result of analyzing in vivo BBB penetration by two-photon microscopy after intravenous administration of transpap-1-FITC and angiopep-2-FITC to a mouse for 5 minutes in order to compare and analyze the BBB penetration of transpap-1 and angiopep-2 according to the present application.

[0038] Fig. 5b is a result of analyzing in vivo BBB penetration by two-photon microscopy after intravenous administration of Transitive Peptide-1-FITC and angiopep-2-FITC 10 to mice for 10 minutes in Fig. 5a.

[0039] Fig. 5c is a result of analyzing in vivo BBB penetration by two-photon microscopy after intravenous administration of Transitive Peptide-1-FITC and angiopep-2-FITC 10 to mice for 30 minutes in Fig. 5a.

[0040] Fig. 5d is a result of analyzing in vivo BBB penetration by two-photon microscopy after intravenous administration of Transitive Peptide-1-FITC and angiopep-2-FITC 10 to mice for 60 minutes in Fig. 5a.

[0041] Fig. 5e is a result of analyzing in vivo BBB penetration by two-photon microscopy after intravenous administration of Transitive Peptide-1-FITC and angiopep-2-FITC 10 to mice for 90 minutes in Fig. 5a.

[0042] Fig. 5f is a graph showing quantification of the imaging results of Figs. 5a to 5e.

[0043] BEST MODE

[0044] The present application relates to a cell penetrating peptide which can be widely used in research fields, diagnosis and treatment fields of various diseases, etc., and relates to a basic platform peptide structure which can be expanded to an infinite number of designs.

[0045] Hereinafter, the present application will be described in detail.

[0046] The present application provides a cell penetrating peptide composed of amino acids represented by the following [Formula I]

[0047]

[0048]

Formula I

[0049] X1-X2-...X n-1 -X n

[0050] In the Formula I,

[0051] n≥16,

[0052] X2 is His (H) and X6 is Leu,

[0053] ​The amino acid other than X2 and X6 can be any one selected from the group consisting of glycine (Gly, G), histidine (His, H), glutamic acid (Glu, E), arginine (Arg, R), lysine (Lys, K), serine (Ser, S), aspartic acid (Asp, D), tryptophan (Trp, W), valine (Val, V), threonine (Thr, T), alanine (Ala, A), asparagine (Asn, N), tyrosine (Tyr, Y).

[0054] The "cell permeability" in the present application refers to the ability or property of a peptide to penetrate a cell (membrane) and to penetrate into the cell.

[0055] The "peptide" in the present application is a polymer of amino acids, and the form in which a small number of amino acids are linked is generally referred to as a peptide, and the form in which a plurality of amino acids are linked is referred to as a protein. In such a peptide and protein structure, the linkage between the amino acids consists of an amide bond or a peptide bond. The peptide bond is a bond formed in the form of -CO-NH- by removing water (H2O) between a carboxyl group (-COOH) and an amino group (-NH2).

[0056] In the present application, the cell penetrating peptide represented by the [general formula I] has histidine at the 2nd amino acid from the N-terminus and leucine at the 6th amino acid, and includes 1 to 16 consecutive amino acid sequences, and various amino acids capable of improving the effect of the cell penetrating peptide can be further added to the C-terminus.

[0057] More specifically, the cell penetrating peptide according to the present application can consist of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12, but is not limited thereto. At this time, the cell penetrating peptide can include an amino acid sequence having greater than or equal to 70%, preferably greater than or equal to 80%, further preferably greater than or equal to 90%, most preferably greater than or equal to 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology with the amino acid sequence shown in SEQ ID NO: 1 to 12, respectively.

[0058] In the present application, the cell type that the cell penetrating peptide can penetrate can be any one selected from the group consisting of brain endothelial cells, cancer cells, blood cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells (iPSCs), neural stem cells (NSCs), T cells, B cells, natural killer cells (NK cells), macrophages, microglia, neurons, astrocytes, and muscle cells, but is not limited thereto.

[0059] The peptides of the present application can be prepared by a conventional synthesis method or preparation method known to one of ordinary skill in the art so that the purity of each peptide is at least 90%, for example, can be directly synthesized or purchased after being prepared by a peptide preparation company. The peptides can be prepared by a conventional synthesis method or preparation method known to one of ordinary skill in the art, in the form of D-form or L-form, a peptide in which a partial sequence consists of D-form or L-form, or a racemate thereof. In addition, a conventional modification known in the art other than this can be performed to improve the stability of the peptide. The present application preferably synthesizes the peptide by a polypeptide solid phase synthesis method, but the peptide synthesis method and conditions as described above are not limited thereto.

[0060] The present inventors designed and synthesized cell penetrating peptides having various sequences and lengths based on the existing research on the sequence and structure of the existing known artificial cell penetrating peptide or human cell penetrating domain. As a result, a cell penetrating peptide consisting of the amino acid sequence of SEQ ID NO: 1 having excellent cell penetrating ability and mass transfer effect was found, and was named "transformation peptide-1".

[0061] In addition, based on the three-dimensional structure of the transformation peptide-1 peptide, it was expected that the amino acids at positions 2 and 6 of the amino acid sequence of SEQ ID NO: 1 have an important influence on cell penetration.

[0062] Based on this assumption, the present inventors compared the cell penetrating ability by synthesizing various peptides in which the amino acids at positions 2 and / or 6 were substituted or the amino acids at other positions were substituted, thereby confirming the above assumption.

[0063] More specifically, in an embodiment of the present application, after synthesizing 12 cell penetrating peptides corresponding to the platform peptide structure according to the present application, the in vitro cell penetration results of the verification thereof showed that the 12 cell penetrating peptides satisfying the platform peptide structure according to the present application all showed excellent cell penetration (Reference Example 2).

[0064] In another embodiment of the present application, after synthesizing a single mutant peptide or a double mutant peptide in which the amino acids at positions 2 and / or 6 from the N-terminus were substituted, in vitro cell penetration analysis was performed. As a result, when the amino acids at positions 2 and 6 were substituted alone or simultaneously, the cell penetration of the peptide was significantly reduced, thus specifically confirming that the amino acids at the two positions are very important for the function of the cell penetrating peptide according to the present application (Reference Example 3).

[0065] In another embodiment of the present application, to verify the cell penetration and mass transfer effect of the 12 peptides according to the present application in vivo, a fluorescent protein GFP as a delivery cargo was bound to the peptide having the amino acid sequence of SEQ ID NO: 1 to thereby prepare a cell penetrating peptide-GFP (Reference Example 4). Then, GFP as a negative control group and the cell penetrating peptide-GFP were administered to mice through the tail vein, respectively, and 24 hours later, the brain cortex and hippocampus tissue sections were prepared from the brains of the mice, and IHC analysis was performed using the sections, and the results of the fluorescence images and the quantification thereof showed that the cell penetrating peptide according to the present application also has excellent cell penetration in vivo, and it can effectively deliver a substance into a cell as a carrier (Reference Example 5).

[0066] In another embodiment of the present application, the cell penetration ability of the cell penetrating peptides known in the art and the peptides according to the present application were compared. To this end, angiopep-2-FITC was prepared using the known cell penetrating peptide angiopep-2, and in vitro cell penetration analysis and in vivo BBB penetration analysis experiments were performed using the above-mentioned Transpeptide-1-FITC, respectively. As a result, it was confirmed that the cell penetrating peptide according to the present application has significantly higher cell penetration and excellent in vivo activity and mass transfer efficiency compared to angiopep-2 (Reference Example 6).

[0067] As can be seen from the results of the above-described embodiments, the peptide according to the present application can be used as a carrier for introducing any substance bound to the peptide into a cell.

[0068] To this end, another aspect of the present application provides a complex including the cell penetrating peptide and a biologically active substance.

[0069] In the present application, the complex includes a simple mixture of the peptide and the substance, a complex formed by mixing the peptide and the substance, or a complex formed by chemical linkage or conjugation of the peptide and the substance. In addition, the complex can be linked in the form of physical linkage, chemical linkage, covalent linkage, non-covalent linkage, self-assembly linkage, or fusion or merging using a medium.

[0070] In addition, the complex can be a complex in which the peptide and the biologically active substance are expressed in a state of being fused to each other. For example, when the peptide and a gene expressing a biologically active substance are inserted into one vector, and the organism is transformed with the vector to express the gene inserted into the vector, the peptide and the biologically active substance can be expressed as a fusion protein. When expressed as a fusion protein, any linker can be included between the peptide and the biologically active substance.

[0071] In addition, in the complex according to the present application, the cell penetrating peptide can include a single or a plurality of conjugated forms for efficient delivery of the bioactive substance into the cell, and the number of conjugated cell penetrating peptides can be easily selected or adjusted by those skilled in the art according to the bioactive substance to be delivered.

[0072] In the present application, the bioactive substance forming a complex by conjugation with the cell penetrating peptide preferably means a "substance having biological or pharmaceutical activity", which means a substance penetrating into the cell (cytoplasm or nucleus) to participate in the modulation of physiological activity or to express a pharmacological effect or a substance having biological activity at each site in the body such as the cytoplasm, tissue, interstitium, blood, etc. after being transported to exert an effect. For example, it can be at least one selected from the group consisting of a chemical compound, a protein, a glycoprotein, a peptide, an antibody, an enzyme, a nucleic acid, a hormone, a cytokine, a transcription factor, a toxin, a carbohydrate, a lipid, a glycolipid, a natural product, a semi-synthetic drug, a drug, a microparticle, a nanoparticle, a liposome, a virus, a quantum dot, and a fluorescent dye, but is not limited thereto.

[0073] The nuclease can be selected from the group consisting of CAS9, CAS12, CAS13, CAS14, CAS variants, Cfp1 (CxxC-finger protein-1), ZEN (Zinc-finger nuclease), and TALEN (Transcription activator-like effector nuclease), but is not limited thereto.

[0074] The nucleic acid can be selected from the group consisting of DNA, RNA, antisense oligonucleotide (ASO), miRNA, small interfering RNA (siRNA), aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino, and can further include, but is not limited to, decoy DNA, plasmid, shRNA, antisense RNA, oligoribonucleotide, or transport RNA.

[0075] The drug can be selected from the group consisting of a chemical drug, a biological drug, a nucleic acid drug, a peptide drug, a protein drug, a natural product drug, a hormone, a contrast agent, and an antibody, but is not limited thereto.

[0076] The "biological drug" refers to (original) biological agents, biogenerics, biobetters, biosuperiors, and various biological drugs. The biological drug refers to any drug prepared, secreted, or semi-synthesized from a biological source, which includes, but is not limited to, vaccines, blood agents, antigens, cell agents, gene therapy drugs, stem cells, etc.

[0077] The nanoparticle can be selected from the group consisting of iron oxide, gold, carbon nanotube, and magnetic beads, but is not limited thereto.

[0078] Another aspect of the present application provides a composition for mass transfer, which includes the complex as an active ingredient.

[0079] The composition for mass transfer can be used to deliver a biologically active substance to an in vivo tissue or blood, or to promote cell permeability. The composition can be delivered through cells constituting a living tissue or connections between cells, but the delivery method is not limited.

[0080] The living tissue refers to at least one of epithelial tissue, muscle tissue, neural tissue, and connective tissue, each of which can be composed of at least one tissue, and thus can include, but is not limited to, various living organs such as mucosa, skin, brain, lung, liver, kidney, spleen, lung, heart, stomach, large intestine, digestive tract, bladder, ureter, urethra, ovary, testis, genital organ, muscle, blood, blood vessel, lymph vessel, lymph node, thymus, pancreas, adrenal gland, thyroid gland, parathyroid gland, larynx, tonsil, bronchus, and alveolus.

[0081] When it is necessary to deliver the complex to a specific cell, tissue, or organ, the biologically active substance can be complexed by being bound to a single clone antibody (mAb) and a modified form, the single clone antibody being capable of specifically binding to an extracellular portion protein of a ligand or a receptor or a ligand thereof, the ligand being selectively bound to a receptor specifically expressed in a specific cell, tissue, or organ. The binding between the peptide and the biologically active substance can be achieved by indirect connection through cloning techniques using an expression vector at the nucleotide level or by direct connection through chemical or physical covalent or non-covalent bonds of the peptide and the biologically active substance.

[0082] In the present application, when the composition including the complex is used as a pharmaceutical composition, the composition can include the active ingredient in an amount of 0.001 to 50% by weight based on the total weight of the composition.

[0083] In addition to the active ingredient, the composition of the present application can further include at least one active ingredient having the same or similar function.

[0084] In addition to the active ingredients described above, the composition of the present application can be prepared for administration by further including at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can use physiological saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerol, ethanol, liposome, and a mixture of at least one of these components, and antioxidants, buffers, bacteriostatic agents, and other conventional additives can be added as needed. In addition, it can also be formulated as an injection preparation such as an aqueous solution, a suspension, an emulsion, etc., a pill, a capsule, a granule, or a tablet by additionally adding a diluent, a dispersant, a surfactant, a binder, and a lubricant, and can be used by binding a target organ-specific antibody or other ligand to the carrier to specifically act on the target organ. Further, it can be preferably formulated according to each disease or component using a suitable method of the art or a method disclosed in Remington's Pharmaceutical Sciences.

[0085] The composition including the complex as an active ingredient can be administered intravenously, intraperitoneally, intramuscularly, intrathecally, intracerebroventricularly, subcutaneously, intradermally, intranasally, intramucosally, by inhalation, orally, or the like, to be delivered into the body. The administration amount can vary depending on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease of the administration subject.

[0086] Another aspect of the present application provides a method for delivering a substance into a cell, which includes treating a cell with the composition for substance delivery.

[0087] The cell-penetrating peptide having a mass transfer function according to the present application is a very small peptide, and thus can minimize the biological interference with the active substance that can occur.

[0088] Hereinafter, preferred examples are provided to help understanding of the present application. However, the following examples are intended to more easily understand the present application, and the present application is not limited thereto.

[0089] [Examples]

[0090] Example 1. Preparation of a set of cell penetrating peptide candidates

[0091] 1-1. Designing a cell-penetrating peptide candidate group

[0092] The present inventors designed and synthesized cell-penetrating peptides having various sequences and lengths based on the analysis results of various existing cell-penetrating peptides in previous studies, and verified the cell penetration thereof. As a result, a cell-penetrating peptide composed of the amino acid sequence of SEQ ID NO: 1 showing the highest cell penetration ability was found, and was named Transduction Peptide-1.

[0093] Further, the present inventors synthesized 11 peptide variants by substituting one or two amino acids of the Transforming Peptide-1 sequence in order to further discover cell penetrating peptides. At this time, based on the three-dimensional structure of the Transforming Peptide-1 peptide, it was expected that the amino acids at positions 2 and 6 would affect cell penetration, and thus the amino acids at the above two positions were not modified, but a candidate group of cell penetrating peptides was synthesized by modifying the amino acids at the remaining positions. In summary, a total of 12 peptides synthesized in the present application and their sequences are shown in Table 1 below.

[0094] [Table 1]

[0095]

[0096]

[0097] 1-2. Synthesis and isolation and purification of a candidate group of skin

[0098] The present inventors utilized polypeptide solid phase synthesis (SPPS) to synthesize each of the peptides described in Example 1-1 above. The method is an organic synthesis method in which the C-terminus of an N-terminal F-moc-protected amino acid is bound one by one to the N-terminus of a resin. The solvent used for all reactions was dimethylformamide (DMF), and after mixing an amino acid solution at a concentration of 2M, 1 ml of 0.5M N,N-diisopropylcarbodiimide (DIC), and 0.5 ml of 1M cyano(hydroxyl imino)acetic acid ethyl ester (Oxyma), amino acid coupling was performed by reaction in a microwave synthesizer. In addition, the amino acids were prepared by varying the reaction time, temperature, or microwave voltage for each amino acid sequence. At this time, it was necessary to remove the F-moc of the previous amino acid in order to synthesize the next amino acid, and for this purpose, the F-moc was deprotected twice at 80°C using a solution of 80% DMF and 20% piperidine for 2 minutes each time. Between all coupling processes and deprotection processes, a process of alternating washing with DMF and dichloromethane (DCM) was performed 3 times.

[0099] For the peptide synthesized by the above method, a fluorescent substance including a carboxyl group (-COOH) can be linked to the N-terminus of the peptide by a chemical binding method so as to subsequently observe whether it has cell penetration and quantify it. At this time, the fluorescent substance that can be used has FITC (Fluorescein-5-isothiocyanate), Cyanine 3 carboxylic acid, Cyanine 5 carboxylic acid, Cyanine 7 carboxylic acid, etc. Specifically, among the fluorescent substances as described above, the present embodiment intends to link FITC to the synthesized peptide. To this end, first, after synthesizing the last amino acid of the peptide synthesized with a solid-phase resin, FITC: DIC: Oxyma: resin are mixed in a ratio of 2:2.5:4:1 and then the reaction of the resin is performed at room temperature for 2 hours, but a magnetic stirrer is used. Then, when the color of the resin changes from yellow to dark yellow or orange during the synthesis process, a process of washing with DMF and dichloromethane alternately 3 times is performed. Then, in order to separate the peptide / FITC from the copper solid-phase resin, after the resin synthesized in a cleavage solution in which trifluoroacetic acid (TFA): triisopropylchlorosilane (TIS): distilled water are mixed in a ratio of 95:2.5:2.5 is reacted for 2 hours using a magnetic stirrer, the resin is filtered with a cotton filter. The solution in the filtered solution is evaporated under nitrogen, and when a precipitate is formed, it is precipitated with refrigerated diethyl ether. After drying the precipitated peptide / FITC under vacuum, it is dissolved with distilled water and freeze-dried.

[0100] The freeze-dried peptide was dissolved with distilled water or acetonitrile (ACN), etc., and then separated and purified using reverse phase high-performance liquid chromatography (HPLC). At this time, the mobile phase solvent of the HPLC used solvent A (distilled water 99.9%, TFA 0.1%) and solvent B (distilled water 9.9%, acetonitrile 90%, TFA 0.1%). The HPLC mobile phase was initially 90% solvent A and 10% solvent B, and separation was performed while increasing the solvent B to 1% / minimum gradient. Then, after freeze-drying the separated peptide and removing the solvent, it was dissolved in a desired solvent and experiments were performed.

[0101] Example 2. Analysis of cell penetration in vitro

[0102] The inventors performed in vitro cell penetration analysis on 12 peptide candidate groups synthesized through Example 1 above. Specifically, human blood-brain barrier cells (hCMEC / D3) were seeded at 18,000 cells / well in 96-well plates and cultured overnight in EGM (endothelial cell growth medium) at 37°C and CO2 until the cells occupied 80% to 90% of the plate area. Then, 12 FITC-linked candidate peptides prepared through Examples 1-2 above and FITC as a negative control were diluted at 4 μM in the medium to prepare 100 μl per well for processing. After removing the culture medium from the plate, the peptides were diluted and treated with the pre-prepared solution, and then cultured at 37°C and CO2 for 2 hours. Then, the solution for treating the peptides in each well was removed by soaking, 100 μl of EGM was added and tapped 5 to 6 times, and the process of aspiration and removal was repeated twice. Then, Hoechst 33342 was diluted 1:5000 with EGM, and 100 μl was added to each well. The cells were incubated at 37°C and CO2 for 30 minutes. After incubation, DAPI and GFP fluorescence images were read using a Cytation 5 device, and cell nuclei were delineated using DAPI through image processing. The FITC value at 20 μM around the cell nucleus was then measured and divided by DAPI to obtain the average FITC value. The FITC experimental group was then converted to a negative control group, and the penetration into blood-brain barrier cells was calculated.

[0103] The results are as follows Figure 1 As shown, when each peptide linked to FITC was treated at a concentration of 4 μM, the FITC fluorescence confirmed that all 12 peptides had cell-penetrating properties.

[0104] Example 3. Verification of amino acid positions important for cell penetration

[0105] As described in Example 1 above, the inventors anticipated that the amino acid residues at positions 2 (AA2) and 6 (AA6) in the cell-penetrating peptide-1 of the transforming peptide would have an important influence on cell permeability. The following experiments were conducted to verify this conjecture.

[0106] Therefore, as shown in Table 2 below, single mutant peptides were synthesized in which the histidine (H) at position 2 or the leucine (L) at position 6 of the transforming peptide-1 was replaced by alanine (A) or arginine (R) or valine (V), respectively, and their cell permeability was analyzed by the same method as in Example 2 above.

[0107] [Table 2]

[0108] No. Substance name Mutation 1 #1 AA2 H->A 2 #2 AA6 L->A 3 #3 AA2 H->R 4 #4 AA6 L->V

[0109] As a result, as shown in FIG. 2a, the cell penetration of the four variant peptides in which the second or sixth amino acid residue was varied was significantly reduced compared to the conversion peptide-1 which was confirmed to have cell penetration by Example 2 described above.

[0110] In addition, in order to further confirm the importance of the two amino acid residues, the peptide in which both the second and sixth amino acid residues were substituted with alanine was synthesized as shown in Table 3, and among the peptides 2, 3, and 4 of Table 1 which were confirmed to have cell penetration by Example 1 described above, variant peptides in which double mutations were induced by substituting the second or sixth amino acid with alanine were synthesized, and the same experiment as described above was performed.

[0111] [Table 3]

[0112] No. Substance name Mutation 1 #5 AA2 H->A / AA6 L->A 2 #6 AA2 H->A / AA9 D->A 3 #7 AA2 H->A / AA10 E->A 4 #8 AA2 H->A / AA12 S->A 5 #9 AA6 L->A / AA9 D->A 6 #10 AA6 L->A / AA10 E->A 7 #11 AA6 L->A / AA12 S->A

[0113] As a result, as shown in FIG. 2b, all of the seven variant peptides in which double mutations were induced showed significantly reduced cell penetration. From the above results, it can be confirmed that the histidine at the second position and the leucine at the sixth position are important residues that determine cell penetration in the cell penetrating peptide of the present application.

[0114] Example 4. Preparation of cell penetrating peptides bound to the fluorescent protein GFP

[0115] The present inventors, in order to image the delivery of the cell penetrating peptide according to the present application to in vivo tissues and verify the function thereof as a cargo carrier in the following in vivo cell penetration analysis, intended to prepare a cell penetrating peptide-GFP by conjugating a fluorescent protein GFP (Green Fluorescence Protein) and the cell penetrating peptide of the present application. GFP is a typical fluorescent protein having a size of 27 kDa, an excitation peak at 395 nm and 475 nm, and an emission peak at 509 nm.

[0116] Specifically, in order to prepare a cell penetrating-GFP, the conversion peptide-1 peptide sequence of SEQ ID NO: 1 was inserted into the C-terminus of GFP using a polynucleotide enzyme, and primers capable of binding to the N-terminus and C-terminus were designed using the same as a template. After amplifying the same using PCR, it was inserted into a pET28a expression vector, thereby preparing a recombinant expression vector of the cell penetrating peptide-GFP protein. After transforming E. coli BL21 (DE3) with the recombinant expression vector, the E. coli was cultured until the O.D. value was 0.5, and then IPTG was added at a concentration of 1 mM to induce the expression of the cell penetrating peptide-GFP. Then, the expression level of the protein was confirmed by performing SDS-PAGE, and the protein was separated and purified using His-tag affinity chromatography.

[0117] Example 5. Analysis of cell penetration in vivo

[0118] The present inventors verified the cell penetration of the cell penetrating peptides according to the present application in vivo.

[0119] To this end, among the 12 peptides, the following experiment was performed using Antennapedia-1 as a representative. Specifically, the cell penetrating peptide-GFP prepared by the method described in Example 4 above or GFP as a negative control group was diluted in PBS at a concentration of 500 uM, and 100 ul was injected through the tail vein (i.v. injection) of C57BL / 6 mice. After 24 hours, the mice were anesthetized by intraperitoneal injection of Zoletil at a dose of 0.625 ml / kg, and a toe-pinch test was performed to confirm complete anesthesia. Then, the blood in the body was removed by atrial perfusion at a rate of 3 ml / min for 30 ml of physiological saline, and the tissue was fixed by perfusion of 30 ml of a 4% paraformaldehyde solution at a rate of 3 ml / min. After removing the brain from the skull of the fixed mouse, post-fixation was performed in a 4% paraformaldehyde solution at 4°C for 24 hours. Then, to prevent cell damage during preparation of tissue sections, the brain tissue was transferred to a 30% sucrose solution and the solution was replaced in the tissue at 4°C for 48 hours. After removing the sucrose solution remaining in the brain tissue, the tissue was rapidly frozen using OCT embedding (Optimal cutting temperature compound), and then a tissue section of 50 um was prepared using a cryomicrotome.

[0120] To perform immunohistochemistry (IHC) for the GFP protein using the tissue section prepared by the above method, first, the tissue section was soaked in a 100% methanol solution at 4°C for 10 minutes, and then washed with PBS twice for 5 minutes each. After washing, the tissue was covered with blocking solution (CAS blocking solution), and after incubation at room temperature for 1 hour, the blocking solution was removed. Then, to induce antibody reaction, the primary antibody Rabbit anti-GFP was diluted 1:200 in PBS, and after covering the prepared tissue, incubation was performed at 4°C for 24 hours. After washing with PBS 5 times for 3 times, the secondary antibody Goat anti-Rabbit IgG / Alexa488 was diluted 1:200 in PBS, and after covering the tissue where the reaction was terminated, incubation was performed at room temperature, and then washed with PBS twice for 5 minutes each.

[0121] On the other hand, in order to stain the nucleus, the DAPI solution was diluted in PBS at 1:400, and then covered on the tissue, and incubated at room temperature for 10 minutes, and then washed with PBS twice for 5 minutes each time. The tissue after the termination of the immunohistochemistry experiment was wrapped with the fixing solution, and after covering the cover glass, the periphery thereof was sealed with nail polish and dried for 30 minutes, and then used for imaging. For the brain tissue fixed on the cover glass, the delivered GFP and the nucleus were imaged using a confocal microscope, and the tissue image for analysis was acquired using a 20X lens in a range capable of including the cerebral cortex and the hippocampus. In addition, the image acquired using the confocal microscope was photographed in a resolution of 0.38um x 0.38um x 2.99um (width x height x depth) in a range of 2.5mm x 2.5mm x 40um (Field of view, FOV). By cropping the lost part of the image in the FOV range and performing a pre-processing capable of increasing the signal-to-noise ratio (SNR) through a Gaussian filter, quantification of the acquired image was performed. In order to quantify the GFP of the cerebral cortex and the hippocampus region using the pre-processed image, the cortex region and the hippocampus region were divided based on the cells and the part stained by DAPI, and thus, after preparing an ATLAS of the cortex region, the hippocampus region within the tissue, the fluorescence intensity of GFP was detected to show the degree of immunoreaction.

[0122] As a result, as shown in the result of FIG. 3a, it was confirmed that green fluorescence was not observed in the cerebral cortex and hippocampus tissue of the control group in which only GFP was injected without using the peptide, and in contrast, green fluorescence was clearly observed in the cerebral cortex and hippocampus tissue when the cell penetrating peptide-GFP was injected compared to the control group. It was also confirmed through the quantification result of FIG. 3b that higher fluorescence was detected when the cell penetrating peptide-GFP was injected than when only GFP was injected, for example, about 2.2 times (p=0.009) in the cerebral cortex and about 1.78 times (p=0.009) in the hippocampus. It was confirmed through the above results that the cell penetrating peptide according to the present application also has excellent cell permeability in vivo, and it can effectively deliver cargo to cells as a mass transfer carrier.

[0123] Example 6. Comparison of effects with existing cell penetrating peptides

[0124] The present inventors intended to compare the degree of cell penetration of 12 cell penetrating peptides according to the present application and a cell penetrating peptide known in the related art. To this end, the following cell penetration ability and in vivo blood brain barrier (BBB) penetration analysis experiments were respectively performed using angiopep-2 as the known cell penetrating peptide. The angiopep-2 peptide consists of a 19-amino acid sequence, and it is known to be introduced into cells by binding to low-density lipoprotein receptor-related protein 1 (LRP-1).

[0125] 6-1. Comparative analysis of cell penetration effect by flow cytometry

[0126] First, for the transition peptide-1-FITC and angiopep-2-FITC prepared by the above Example 1-2, the amount of inflow of fluorescent substance inside a single cell was confirmed and quantified by flow cytometry. Specifically, the hCMEC / D3 cell line was inoculated at 15000 cells / well using EGM medium containing 2% BCS (bovine calf serum), and after 24 hours, the cells were treated with the two peptides at a concentration of 10 uM, respectively, and then incubated for 2 hours at 37°C under CO2 conditions. Then, the cells were washed with 1X PBS, and 1.1% TE (Trypsinase-EDTA) was added to the cells for 3 minutes to allow the cells to fall from the bottom of the plate, and then about 3 times the amount of EGM cell culture medium was added, and centrifuged at 100 rpm for 5 minutes. Then, after aspirating the supernatant, the cells were resuspended in 250 μl of 1x PBS and transferred to a BD Falcon 12x75mm tube with a cell strainer cap, and the amount of inflow of each peptide according to the fluorescent substance was analyzed using a flow cytometer.

[0127] The results are shown in Table 1. Figure 4 As shown in Table 1, when treated with the transition peptide-1 peptide, a significantly higher fluorescence intensity was shown than when treated with the angiopep-2-FITC peptide (p<0.0001). Thus, it was confirmed that the transition peptide-1 peptide according to the present application has a significantly higher cell penetration than the angiopep-2 peptide.

[0128] 6-2. Comparative analysis of in vivo blood-brain barrier penetration

[0129] Then, in order to compare the in vivo BBB penetration of the transition peptide-1 and angiopep-2 according to the present application, experiments were performed by the following method.

[0130] Specifically, for in vivo live imaging using two-photon microscopy, a skull window setting surgery was performed on a live mouse (C57BL / 6). Before making the skull window, the mouse was anesthetized by 3% isoflurane, and the body temperature of the mouse was maintained at 36.5 to 37.5°C using a heating pad. After the induction of anesthesia, the anesthesia was maintained by adjusting the concentration of isoflurane to 1.5%. In addition, to confirm the degree of anesthesia maintenance, the heart rate and SpO2 were confirmed in real time. Then, a craniotomy was performed using a dental drill so as to have a diameter of 3 mm at the skull coordinates of ML, +2.5 mm, AP, -1.5 mm. After the skull was removed by the craniotomy, a cover glass having a diameter of 4 mm was overlaid, and then was bonded using a cyanoacrylate adhesive. Then, under the two-photon microscope, a head frame capable of fixing the head of the mouse was bonded so that the cover glass was located at the center. After the one bonding of the cover glass and the frame using the adhesive, all of the exposed skull was covered from the boundary of the cover glass using a dental resin so as to be capable of imaging by the two bonding and using a water immersion lens. After the surgery, 5 mg / kg of enrofloxacin and meloxicam were injected to relieve inflammation caused by the surgery.

[0131] After the skull window surgery according to the above procedure, a recovery period of 4 to 6 weeks was performed, and then the cover glass of the skull window was wiped to remove the suspended matter using secondary distilled water and 70% alcohol so that only pure distilled water was present between the lens and the cover glass at the time of the experiment. Then, after the mouse was anesthetized using 2% isoflurane, the head frame was transferred to a stereotaxic frame, the anesthesia was maintained using 1.5% isoflurane, and the body temperature was constantly maintained at 36.5 to 37.5°C, and then a tube was fixed to the tail vein and was fixed. In order to use a water immersion lens (25X, NA: 0.9), distilled water was injected into the washed skull window and was focused, and then Texas red conjugated dextran was injected at a dose of 1.5 ml / kg (body weight, concentration: 5% (w / v)) through the tail vein tube.

[0132] Then, the imaging range (FOV) is set so that the pial artery, penetrating arteriole, venule, and pial vein can all be included, and vascular structure imaging is performed. As described above, the pial vessel is imaged for the vascular structure image (1) at a depth of about 350 to 400 um. Then, the image parameters are adjusted for in vivo real-time imaging (2) so that the visual resolution is 1 minute, and a range of 354 um x 354 um is imaged at a resolution of 512 x 512. The resolution of the z-axis is 2 um and 75 images are obtained, i.e., a thickness of 150 um is set as a thickness of 50 to 200 um in depth excluding the pial vessel. After the settings for performing the in vivo real-time imaging (2) are performed, 15 mg / kg of the transition peptide 1-FITC or angiopep-2-FITC is injected through the tail vein, and then imaging is performed for 90 minutes using the same image parameters as the settings.

[0133] After the vascular structure image (1) and the in vivo real-time imaging image (2) obtained by the above-described method are reconstructed into three-dimensional matrices, the in vivo real-time imaging matrix is registered to the vascular structure image matrix. The registration method in principle uses linear registration, and the rigid body and similarity parameters are selectively used according to movement artifacts at the time of obtaining the image. In the registered real-time imaging matrix, the internal region of the blood vessel is defined as the intravascular region and the external region is defined as the extravascular region using the blood vessel internal region binarized with 70 kDa-texas red dextran.

[0134] Binarization can be performed using imageJ and matlab. imageJ uses CLAHE (local contrast enhanced thresholding), and matlab performs it by using an algorithm using Mexican hat, otsu, and local contrast. After the internal and external regions of the blood vessel are determined, the coordinates thereof are applied to the coordinates of the real-time imaging matrix, and the change of the transition peptide 1-FITC or angiopep-2-FITC in the intravascular / extravascular region is observed.

[0135] In addition, in order to quantify the results observed through the above-described imaging images, the intensity average of the detected initial intravascular region is set to 100% and the intensity average of the initial extravascular region is set to 0%, and the voxel values of the matrix present on all clock domains are normalized, thereby mapping the percentage to the in vivo real-time imaging matrix. The normalized intensity value is obtained by the following equation. At this time, in the following equation, averaged Intra Intensity0 represents the intensity average of the initial intravascular region, and averaged extra Intensity0 represents the intensity average of the initial extravascular region.

[0136]

[0137] As a result, as shown in FIGS. 5a to 5f, it was confirmed that when the transition peptide-1-FITC was injected, a longer and higher level of fluorescence was observed in the intravascular region than when the angiopep-2-FITC was injected, thereby confirming higher in vivo stability. In addition, when the transition peptide-1-FITC was injected, a greater amount was delivered to the extravascular region than when the angiopep-2-FITC was injected. In summary, it was confirmed that the transition peptide-1 according to the present application has more excellent in vivo stability and delivery efficiency than the angiopep-2 peptide.

[0138] The above description of the present application is exemplary, and those skilled in the art to which the present application pertains can understand that it can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present application. Therefore, it should be understood that the above-described embodiments are exemplary rather than limiting in all aspects.

[0139] Industrial applicability

[0140] The new cell penetrating peptide according to the present application has excellent cell penetrating ability and mass transfer effect, and can effectively deliver substances having various biological activities to cells, tissues, and the like in vivo, and thus is expected to be widely used in research fields, diagnosis or treatment fields of various diseases, and the like.

Claims

1.A cell penetrating peptide consisting of amino acids represented by the following [Formula 1]: [Formula I] wherein, X 1 is alanine, X 2 is histidine, X 3 is glycine, X 4 is glycine, X 5 is glycine, X 6 is leucine, X 7 is glycine, X 8 is glycine, X 9 is glycine, X 10 is glycine, X 11 is glycine, and X 12 is glycine. 2.The cell penetrating peptide of claim 1, wherein: the cell is selected from the group consisting of brain endothelial cells, cancer cells, blood cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells, neural stem cells, T cells, B cells, natural killer cells, macrophages, microglia, neurons, astrocytes, and muscle cells. X1-X2-...X n-1 -X n 3.A complex comprising the cell penetrating peptide of claim 1 or 2 and a bioactive substance. n≥16， 4.The complex of claim 3, wherein: the bioactive substance is at least one selected from the group consisting of a compound, a protein, a peptide, a nucleic acid, a microparticle, a nanoparticle, a liposome, a virus, and a quantum dot. 5.The complex of claim 4, wherein: the compound is at least one selected from the group consisting of a carbohydrate, a lipid, a semisynthetic drug, a drug, and a fluorescent dye. 6.The complex of claim 4, wherein: the protein is at least one selected from the group consisting of a glycoprotein, an antibody, an enzyme, a nuclease, a hormone, a cytokine, a transcription factor, and a toxin. the nuclease is selected from the group consisting of CAS9, CAS12, CAS13, CAS14, CAS variants, Cfp1, ZEN, and TALEN. the nucleic acid is selected from the group consisting of DNA, RNA, an antisense oligonucleotide, miRNA, small interfering RNA, an aptamer, LNA, PNA, and morpholino. 9.A composition for mass transfer comprising the complex of claim 3. ​ ​ ​ ​ ​ ​ ​ ​ 7. The composite of claim 6, wherein: ​ 8. The composite of claim 4, wherein: ​ ​

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