Novel peptide and micelle comprising same

Abstract

The present invention relates to a novel peptide, a micelle containing the same, and a drug delivery complex containing said micelle. The micelle containing the novel peptide of the present invention can form a complex with a drug such as siRNA, and said complex has excellent cell permeability so that the drug can be effectively delivered into a cell or tissue, so said micelle can be utilized as a drug delivery vehicle.

Description

Technology Field

[0001] The present invention relates to a novel peptide, a micelle comprising the same, and a drug delivery complex comprising said micelle. Background Technology

[0002] Existing drug development technologies primarily utilized small molecule compounds or antibodies, but since these were applied to generated proteins, they had the disadvantage of requiring a long time to derive new drug candidates and having limited target proteins.

[0003] To overcome the aforementioned drawbacks, nucleic acid therapeutics utilizing RNA interference technology are attracting attention in new drug development. Nucleic acid therapeutics using RNA interference technology act on mRNA to inhibit the expression of specific proteins, offering the advantages of a short development time for identifying new drug candidates and the ability to multi-target.

[0004] However, siRNA, which is primarily used as a nucleic acid therapeutic agent utilizing RNA interference technology, has a disadvantage of having very low cell membrane permeability due to its negative charge, so it must be delivered into the cell using a specific carrier.

[0005] Recently, N-Acetylgalactosamine (GalNAc) has been utilized as a carrier for siRNA, but it is mostly used as a treatment for liver-related diseases because it has a high affinity for ASGPR, a specific cell surface protein expressed in liver cells.

[0006] Accordingly, the inventors synthesized a novel peptide that exhibits high cell permeability without exhibiting cytotoxicity, and confirmed that the peptide forms micelles by self-assembly and that the micelles, which form a stable complex with siRNA, have excellent cell internalization and endosome escape capabilities, thereby completing the present invention. The problem to be solved

[0007] One aspect provides a peptide comprising the amino acid sequence of SEQ ID NO. 1.

[0008] Another aspect provides a micelle comprising the above-mentioned peptide.

[0009] Another aspect provides a drug delivery complex comprising the above micelle; and a target drug.

[0010] Another aspect provides a drug delivery composition comprising the above micelle.

[0011] Another aspect provides a pharmaceutical composition for the prevention or treatment of proliferative diseases, comprising the above micelles; and a desired drug. means of solving the problem

[0012] One aspect is to provide a peptide comprising the amino acid sequence of SEQ ID NO. 1.

[0013] The above peptide may be for forming micelles and / or drug delivery vehicles. Specifically, the above peptide may form micelles and / or drug delivery vehicles by self-assembly.

[0014] In one embodiment, the peptide may include and / or be composed of the amino acid sequence of SEQ ID NO. 1, specifically, it may include and / or be composed of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with the amino acid sequence of SEQ ID NO. 1, and may include, without limitation, any amino acid sequence that exhibits substantially the same or corresponding efficacy as the peptide.

[0015] The term "homology" in this specification refers to the degree of similarity between a base sequence or amino acid sequence encoding a protein, and when homology is sufficiently high, the expression product of the gene may have the same or similar activity. Additionally, homology may be expressed as a percentage according to the degree of correspondence with a given amino acid sequence or base sequence. In this specification, a homologous sequence having the same or similar activity as a given amino acid sequence or nucleotide sequence is indicated as "% homology." For example, this can be verified by using standard software, specifically BLAST 2.0, to calculate parameters such as score, identity, and similarity, or by comparing sequences through hybridization experiments performed under defined stringent conditions, and the defined appropriate hybridization conditions may be determined by methods well known to those skilled in the art within the scope of the art.

[0016] The above peptide may additionally include histidine at one or more of the amino terminus and carboxyl terminus, and specifically, one or more histidines may additionally be connected to one or more of the amino terminus and carboxyl terminus of the above peptide.

[0017] The above peptide may additionally include 1 to 20 histids, and specifically, may additionally include 1 to 20, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 2 to 20, 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 3 to 20, 3 to 15, 3 to 12, 3 to 10, 3 to 8, or 3 to 6 histids.

[0018] In one embodiment, the peptide may additionally comprise 1 to 10 histids at the amino terminus, and specifically, may additionally comprise 1 to 10, 1 to 8, 1 to 7, 1 to 6, 2 to 10, 2 to 8, 2 to 7, 2 to 6, 3 to 6, 3 to 10, 3 to 8, 3 to 7, or 3 to 6 histids.

[0019] In one embodiment, the peptide may additionally comprise 1 to 10 histids at the carboxyl terminus, specifically 1 to 10, 1 to 8, 1 to 7, 1 to 6, 2 to 10, 2 to 8, 2 to 7, 2 to 6, 3 to 6, 3 to 10, 3 to 8, 3 to 7, or 3 to 6 histids.

[0020] In one embodiment, the peptide may additionally include 1 to 10 histids at each amino terminus and carboxyl terminus, and specifically, may additionally include 1 to 10, 1 to 8, 1 to 7, 1 to 6, 2 to 10, 2 to 8, 2 to 7, 2 to 6, 3 to 6, 3 to 10, 3 to 8, 3 to 7, or 3 to 6 histids.

[0021] The above peptide may further comprise a polyhistidine peptide composed of one or two to ten histidines at one or more of the amino terminus and the carboxyl terminus.

[0022] In one embodiment, the peptide comprises 1) a first peptide composed of the amino acid sequence of SEQ ID NO. 1 and 2) a second peptide composed of 1 to 10 histids, and / or is composed thereof, wherein the second peptide may be a peptide directly or indirectly connected to the amino terminus or carboxyl terminus of the first peptide through a linker. Additionally, the second peptide may comprise and / or be composed of 1 to 10, 1 to 8, 1 to 7, 1 to 6, 2 to 10, 2 to 8, 2 to 7, 2 to 6, 3 to 6, 3 to 10, 3 to 8, 3 to 7, or 3 to 6 histids.

[0023] In one embodiment, the peptide may comprise and / or be composed of 1) a first peptide composed of the amino acid sequence of SEQ ID NO. 1, 2) a second peptide composed of 1 to 10 histids, and 3) a third peptide composed of 1 to 10 histids, wherein the second peptide is directly or indirectly connected to the amino terminus of the first peptide through a linker, and the third peptide is directly or indirectly connected to the carboxyl terminus of the first peptide through a linker. Additionally, the second peptide may include and / or be composed of 1 to 10, 1 to 8, 1 to 7, 1 to 6, 2 to 10, 2 to 8, 2 to 7, 2 to 6, 3 to 6, 3 to 10, 3 to 8, 3 to 7, or 3 to 6 histids. Additionally, the third peptide may include and / or be composed of 1 to 10, 1 to 8, 1 to 7, 1 to 6, 2 to 10, 2 to 8, 2 to 7, 2 to 6, 3 to 6, 3 to 10, 3 to 8, 3 to 7, or 3 to 6 histids.

[0024] In one embodiment, the peptide may include and / or be composed of any one of the amino acid sequences of SEQ ID NOs 2 to 4, specifically, it may include and / or be composed of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with any one of the amino acid sequences of SEQ ID NOs 2 to 4, and may include, without limitation, any amino acid sequence that exhibits substantially the same or corresponding efficacy as the peptide.

[0025] The above peptide may have a conservative substitution of 1 to 5 amino acids in any one or more of the amino acid sequences of SEQ ID NOs 1 to 4, but is not limited thereto.

[0026] The term “conservative substitution” in this specification means substituting one amino acid with another amino acid having similar structural and / or chemical properties. The peptide may have, for example, one or more conservative substitutions while still retaining the biological activity of the natural or unmodified peptide. Such amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In addition, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains. Amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine, while amino acids with uncharged side chains can be further classified into nonpolar or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, and proline; polar amino acids may include serine, threonine, cysteine, asparagine, and glutamine. Conservative substitution with amino acids having similar properties as described above can be expected to exhibit the same or similar activity.

[0027] In one embodiment, the peptide may include the amino acid sequence of SEQ ID NO. 1 two or more times, and specifically, may include the amino acid sequence of SEQ ID NO. 1 two, three, five, seven, or ten times.

[0028] The above peptide may be a cationic peptide or a hydrophilic peptide. Additionally, the above peptide may be a cell-permeable and / or skin-permeable peptide.

[0029] The term "cell permeability" in this specification means the ability or property of a peptide to penetrate a cell membrane and enter the cell.

[0030] The term "skin permeability" in this specification refers to a peptide that can penetrate the skin regardless of the size or properties of the molecule, can be delivered evenly over the entire skin, and has excellent skin permeability and skin persistence.

[0031] Since the above peptide has cell permeability and / or skin permeability characteristics, the peptide or a carrier such as a micelle containing the same can transport or deliver a drug containing various active substances, such as low molecular weight substances, into a cell or into the skin. Accordingly, the above peptide may be a peptide for drug delivery.

[0032] To obtain chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), modified specificity (e.g., broad spectrum of biological activity), and reduced antigenicity, the above peptide may have a protecting group selectively attached to the N- or C-terminus of the peptide. In one embodiment, the N-terminus of the peptide may be attached to any one protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, and polyethylene glycol (PEG); and / or the C-terminus of the peptide may be bonded to any one of the protecting groups selected from the group consisting of an amino group (-NH2), a tertiary alkyl group, and an azide (-NHNH2). Additionally, the peptide may optionally further include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability.

[0033] The term "stability" in this specification may mean not only in vivo stability, which protects the peptide from attack by protein-cleaving enzymes in vivo, but also storage stability (e.g., room temperature storage stability).

[0034] The above peptide may be prepared by a chemical peptide synthesis method known in the art, for example, the SPSS (Solid Phase Peptide Synthesis) method, or by a process of amplifying the gene encoding the peptide by PCR (polymerase chain reaction) or synthesizing it by a known method and then cloning it into an expression vector for expression, but is not limited thereto. In one embodiment of the present invention, the above peptide may refer to a peptide prepared using cells or an artificially synthesized peptide, but is not limited thereto. For example, the above peptide may be obtained as a recombinant by inserting DNA encoding the peptide into a suitable expression system, or may be artificially synthesized, but is not limited thereto.

[0035] The above peptide may be prepared using human-derived peptides, non-human-derived peptides, or viral peptides, but is not limited thereto.

[0037] Another aspect is to provide a micelle containing the above-mentioned peptide. The same parts as described above apply equally to the micelle.

[0038] The term "micelle" in this specification may refer to a nano-sized particle having a core / shell structure due to the self-assembly characteristics of a peptide.

[0039] The above micelles may be cell-permeable and / or skin-permeable.

[0040] The micelle may be intended for delivering drugs, specifically for delivering any drug to a specific target area (cell, tissue and / or organ).

[0041] In one embodiment, the peptide may additionally include a hydrophobic substance at the amino terminus or carboxyl terminus, specifically at the amino terminus. Additionally, the hydrophobic substance may include one or more hydrophobic moietyes selected from the group consisting of sterols, cholesterol, hydrophobic peptides, and fatty acids. For example, the additionally included hydrophobic substance may be included as a core of the micelle to further increase the stability of the micelle.

[0042] The fatty acid above is a carboxylic acid comprising a saturated or unsaturated aliphatic chain having about 4 or more carbon atoms, and may be, for example, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vacsenic acid, linoleic acid, linoleic acid, linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, etc.

[0043] The above sterols may include animal sterols and derivatives such as cholesterol, cholesteryl chloride, cholesteryl octanoate, cholesteryl nonanoate, cholesteryl oliyl carbonate, cholesteryl isostearyl carbonate, etc., plant sterols and derivatives such as phytosterols, campesterol, sitosterol, stigmasterol, etc., or combinations thereof.

[0044] In one embodiment, the micelle may additionally include one or more of a targeting substance, a labeling substance, and a linker for connecting the targeting substance or the labeling substance.

[0045] The above targeting substance refers to a substance capable of targeting a region (cell, tissue, and / or organ) to which the micelle is to be delivered, and the above targeting substance may include a ligand or a fragment thereof capable of binding to a receptor present in the targeting region; or a receptor or a fragment thereof capable of binding to a ligand present in the above targeting substance. The above targeting substance may include peptides, aptamers, and small molecule compounds, and specifically, may include one or more selected from the group consisting of folic acid (FA), A10, A10-3, DUPA, and ACUPA.

[0046] The above labeling material may refer to a label capable of providing a detectable signal directly or indirectly, and may include, for example, radioactive isotopes, fluorescent molecules, or biotin, and specifically may include fluorescein, phycoerythrin, rhodamine, lissamine, Cy3, Cy5 (Pharmacia), and 6-carboxyfluorescein (6-FAM).

[0047] The above targeting substance and / or labeling substance may be connected through a linker included in the micelle. The linker may refer to a substance used to effectively connect (bind) the targeting / labeling substance to a peptide.

[0048] In one embodiment, the peptide may further comprise a linker for connecting a targeting substance, a labeling substance, or a targeting / labeling substance to an amino terminus or a carboxyl terminus, and specifically may further comprise a linker at the carboxyl terminus.

[0049] In one embodiment, the micelle may further comprise one or more of lipids and biocompatible polymers.

[0050] The above biocompatible polymer may include one or more selected from the group consisting of PLGA (polylactide-co-glycolide), PLA (polylactic acid), PLLA (poly-L-lactic acid), PDLLA (poly-d,l-lactic acid), and PDLA (poly-D-lactic acid), and specifically may include PLGA.

[0051] The above lipids may include one or more selected from the group consisting of phospholipids, PEG-lipids, and ionized lipids.

[0052] The lipids include DSPE (distearoylphosphatidylethanolamine), DSPE-PEG, DSPC (distearoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), POPC (palmitoyloleoylphosphatidylcholine; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), EPC (egg phosphatidylcholine), DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), PE (phosphatidylethanolamine,), DPPE (dipalmitoylphosphatidylethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), It may include one or more selected from the group consisting of DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]), sphingomyelin, and DMG-PEG (1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol), and specifically may include one or more of DSPE and DSPE-PEG.

[0054] Another aspect is to provide a drug delivery complex comprising the micelle; and a target drug. The same parts described above apply equally to the complex.

[0055] The above drug delivery complex may be for intradermal, transdermal, or intracellular drug delivery.

[0056] The above drug delivery complex may be for enhancing cell permeability or skin permeability, and specifically may be for enhancing the cell permeability and / or skin permeability of the above-mentioned target drug.

[0057] The above drug is not particularly limited to active ingredients or physiologically active substances that can be delivered into cells, tissues, or skin and exhibit pharmacological activity that regulates activity within cells, tissues, or skin, and specifically, the above drug may comprise one or more selected from the group consisting of compounds, proteins, peptides, and nucleic acids.

[0058] The above compound may be any one selected from the group consisting of fats, carbohydrates, dyes, photosensitizers, anticancer agents, antibiotics, and low molecular weight compounds, but is not limited thereto.

[0059] The above protein or peptide may be any one selected from the group consisting of enzymes, ligands, hormones, carriers, immunoglobulins, antibodies, structural proteins, motor function peptides, receptors, signal transduction peptides, storage peptides, membrane peptides, transmembrane peptides, internal peptides, external peptides, secretory peptides, viral peptides, native peptides, glycosylated proteins, fragmented proteins, disulfide-binding proteins, recombinant proteins, and chemically modified proteins, but is not limited thereto.

[0060] The above nucleic acid may be any one selected from the group consisting of coding nucleic acid sequences, DNA, mRNA, siRNA, microRNA, plasmid, gene, ASO, gapmer, aptamer, antagomir, agomir, oligonucleotide, and antisense RNA, but is not limited thereto.

[0061] The above drug may be a substance that cannot be introduced into cells, tissues, or skin, or cannot be introduced into cells, tissues, or skin at a useful rate.

[0062] In one embodiment, the drug may be mutually bound to the micelle or the peptide within the micelle to form a conjugate or complex, or mixed with each other without being mutually bound to form a non-covalent conjugate, and is not limited thereto as long as it can improve cell permeability without inhibiting the pharmacological activity of the drug.

[0063] The above drug delivery complex may be formed such that the peptide or micelle containing it and the drug are chemically or physically bonded, e.g., covalently or non-covalently, to rapidly and safely permeate into cells through in vivo or in vitro processing. For example, the complex in which the peptide or micelle containing it and the drug are bound may be introduced directly into the cell without the process of endocytosis, which is a conventional method of intracellular absorption, but is not limited thereto.

[0064] The above drug can electrostatically bind to the above peptide. For example, since the above peptide carries a positive charge, a negatively charged drug can be utilized by electrostatically binding to the above peptide without limitation, as long as the present invention can be applied. Specifically, the above drug can electrostatically bind to the above hydrophilic amino acid residue. For example, since the above hydrophilic amino acid residue carries a positive charge, a negatively charged drug can be utilized by electrostatically binding to the above hydrophilic amino acid residue without limitation, as long as the present invention can be applied.

[0065] In one embodiment, the micelle and the target drug (specifically, nucleic acid, etc.) may be included in the drug delivery complex in a mass ratio (w / w) of 0.1:1 to 20:1, specifically, the mass ratio of the micelle and the target drug (micelle:drug) may be 0.1:1 to 20:1 (w / w), 0.1:1 to 15:1 (w / w), 0.1:1 to 12:1 (w / w), 0.1:1 to 10:1 (w / w), 0.1:1 to 9:1 (w / w), 0.1:1 to 8:1 (w / w), 0.1:1 to 7:1 (w / w), 0.1:1 to 6:1 (w / w), 0.1:1 to 5:1 (w / w), 0.1:1 to 4:1 (w / w), 0.1:1 to 3:1 (w / w), 0.1:1 to 2:1 (w / w), 0.1:1 to 1:1 (w / w), 0.5:1 to 20:1 (w / w), 0.5:1 to 15:1 (w / w), 0.5:1 to 12:1 (w / w), 0.5:1 to 10:1 (w / w), 0.5:1 to 9:1 (w / w), 0.5:1 to 8:1 (w / w), 0.5:1 to 7:1 (w / w), 0.5:1 to 6:1 (w / w), 0.5:1 to 5:1 (w / w), 0.5:1 to 4:1 (w / w), 0.5:1 to 3:1 (w / w), 0.5:1 to 2:1 (w / w), 0.5:1 to 1:1 (w / w), 1:1 to 20:1 (w / w), 1:1 to 15:1 (w / w), 1:1 to 12:1 (w / w), 1:1 to 10:1 (w / w), 1:1 to 9:1 (w / w), 1:1 to 8:1 (w / w), 1:1 to 7:1 (w / w), 1:1 to 6:1 (w / w), 1:1 to 5:1 (w / w), 1:1 to 4:1 (w / w), 1:1 to 3:1 (w / w), 1:1 to 2:1 (w / w), 2:1 to 20:1 (w / w), 2:1 to 15:1 (w / w), 2:1 to 12:1 (w / w), 2:1 to 10:1 (w / w), It may be included in a mass ratio of 2:1 to 9:1 (w / w), 2:1 to 8:1 (w / w), 2:1 to 7:1 (w / w), 2:1 to 6:1 (w / w), 2:1 to 5:1 (w / w), 2:1 to 4:1 (w / w), 2:1 to 3:1 (w / w), 5:1 to 20:1 (w / w), 5:1 to 15:1 (w / w), 5:1 to 15:1 (w / w), 5:1 to 10:1 (w / w), 5:1 to 9:1 (w / w), 5:1 to 8:1 (w / w), 5:1 to 7:1 (w / w), and 5:1 to 6:1 (w / w).

[0066] In one embodiment, the drug delivery complex may be capable of simultaneously delivering two or more different types of drugs into a cell. For example, the drug delivery complex may be capable of simultaneously delivering two or more different types of siRNA into a cell.

[0068] Another aspect is to provide a drug delivery composition comprising the micelles described above. The same parts as described above also apply to the composition.

[0069] In one embodiment, the drug delivery composition may additionally include a target drug.

[0070] The above composition may be a composition for drug delivery into the skin, transdermally, in tissues, or in cells.

[0071] The above composition may be a composition for enhancing cell permeability or a composition for enhancing skin permeability, and specifically, may be a composition for enhancing the cell permeability and / or skin permeability of the above-mentioned target drug.

[0072] In one embodiment, the composition may be for enhancing the cell permeability or skin permeability of the drug, or for delivering the drug into the cell or into the skin.

[0074] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of proliferative diseases, comprising the micelle and a target drug. The same parts described above apply equally to the composition.

[0075] The above proliferative diseases may include one or more selected from the group consisting of neoplasms (including intraepithelial neoplasms), tumors, cancer, leukemia, psoriasis, bone diseases, fibroproliferative disorders, and atherosclerosis, but are not limited thereto.

[0076] The term "cancer" as used in this specification refers to a disease caused by cells having aggressive characteristics of dividing and proliferating beyond normal growth limits, invasive characteristics of infiltrating surrounding tissues, and metastatic characteristics of spreading to other parts of the body, and may be used interchangeably with "malignant tumor."

[0077] The above cancer may be a solid tumor or a blood cancer. The above cancers include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, peritoneal cancer, choriocarcinoma, connective tissue cancer, gastrointestinal cancer, endometrial cancer, esophageal cancer, eye cancer, colon cancer, rectal cancer, kidney cancer, melanoma, stomach cancer, liver cancer, lung cancer (small cell lung cancer, non-small cell lung cancer, adenocarcinoma, squamous carcinoma), colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, respiratory cancer, salivary gland carcinoma, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, urinary system cancer, vulvar cancer, oral cancer, leukemia, multiple myeloma, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, brain tumor, glioblastoma, neuroblastoma, rhabdomyosarcoma, retinoblastoma, head and neck cancer, salivary gland cancer, Hodgkin lymphoma, non-Hodgkin lymphoma. It may include, but is not limited to, one or more selected from the group consisting of mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom macroglobulinemia, post-transplant lymphoproliferative disorder, nevus, Meig syndrome, and lymphoma.

[0078] Furthermore, "treatment of cancer" means inhibiting or preventing the growth of cancer cells or tissues, and this concept includes reducing cancer growth and metastasis compared to when treatment or no treatment is performed, as well as reducing resistance to anticancer drugs to enhance therapeutic effects. The aforementioned metastasis refers to the process of tumor (cancer) cells spreading to distant parts of the body, and "resistance to anticancer drugs" refers to a situation where, when treating cancer patients with anticancer drugs, there is no therapeutic effect from the beginning of treatment, or although there is an initial therapeutic effect, the therapeutic effect is lost during the course of continuous treatment. "Prevention" may refer to any act of inhibiting the occurrence of cancer or delaying its onset through the administration of the aforementioned pharmaceutical composition.

[0079] The above pharmaceutical composition may be used for the prevention or treatment of age-related macular degeneration (wet or dry macular degeneration), connective tissue growth factor-related diseases or disorders (non-limiting examples include keloids, hypertrophic scars, fibrosis, etc.), hair loss diseases, or pigmentation-related disorders. Here, keloids may be burn keloids, posttraumatic keloids, and other types of abnormal proliferation of scar tissue. Non-limiting examples of fibrosis include renal fibrosis, retinal fibrosis, pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, pachydermatosis, or cutaneous fibrosis.

[0080] The above pharmaceutical composition may include a pharmaceutically acceptable carrier. The term "pharmaceuticalally acceptable carrier" may refer to a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the injected compound. Here, "pharmaceuticalally acceptable" means that the target of application (prescription) does not possess toxicity beyond an acceptable level without inhibiting the activity of the active ingredient. Any type of carrier that is commonly used in the relevant technical field and is pharmaceutically acceptable may be used in the above pharmaceutical composition. Non-limiting examples of the above carriers include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. These may be used alone or in a mixture of two or more. The above pharmaceutical composition may be prepared into an oral or parenteral formulation according to the route of administration by conventional methods known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The above pharmaceutical compositions may each be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions according to conventional methods.

[0081] When formulating the above pharmaceutical composition, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, or surfactants, but is not limited thereto.

[0082] When the above pharmaceutical composition is prepared as an oral formulation, it may be prepared in the form of powder, granules, tablets, pills, coated tablets, capsules, liquids, gels, syrups, suspensions, wafers, etc., in accordance with methods known in the art together with a suitable carrier. Examples of pharmaceutically acceptable suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinylpyrrolidone; water; methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate; mineral oil; malt; gelatin; talc; polyols; vegetable oils, etc. In the case of formulation, the formulation may include diluents and / or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants as needed.

[0083] When the above pharmaceutical composition is prepared as a parenteral formulation, it may be formulated in the form of an injectable, transdermal, nasal inhalant, and suppository according to methods known in the art with a suitable carrier. When formulated as an injectable, suitable carriers may include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof; preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose, etc. When formulated as a transdermal formulation, it may be formulated in the form of an ointment, cream, lotion, gel, topical solution, paste, liniment, aerosol, etc. In the case of nasal inhalers, they can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide, and when formulated as suppositories, the base may be Witepsol, Tween 61, polyethylene glycols, cocoa starch, laurin starch, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, and sorbitan fatty acid esters.

[0084] The above pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dose level may be determined based on factors including the severity of the disease, drug activity, patient's age, weight, health, gender, patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and elimination rate, the duration of treatment, drugs combined or used concurrently with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a component known to exhibit a known therapeutic effect. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration.

[0085] The dosage of the above pharmaceutical composition may be determined by a person skilled in the art by taking into consideration the purpose of use, the degree of toxicity of the disease, the patient's age, weight, gender, medical history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered at a dose of about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg per adult, and although the frequency of administration of the composition of the present invention is not particularly limited thereto, it may be administered once a day or administered several times by dividing the dose. The above dosage or frequency of administration does not limit the scope of the present invention in any way.

[0087] Another aspect provides a method for preventing or treating a proliferative disease, comprising the step of administering the above-described pharmaceutical composition to an individual. The same parts as described above also apply to the above method.

[0088] As used in this specification, the term "individual" may include, without limitation, mammals including dogs, cats, rats, livestock, and humans, birds, reptiles, farmed fish, etc., that have or are at risk of developing a proliferative disease, and said individual may exclude humans.

[0089] The above pharmaceutical composition may be administered as a single or multiple doses in pharmaceutically effective amounts. In this case, the composition may be administered in the form of a liquid, powder, aerosol, injection, intravenous fluid (Ringer), capsule, pill, tablet, suppository, or patch. The route of administration of the above pharmaceutical composition may be any general route as long as it can reach the target tissue.

[0090] The above pharmaceutical composition is not particularly limited thereto, but may be administered via routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, nasal administration, pulmonary administration, or rectal administration, depending on the purpose. However, when administered orally, it may be administered in an unformulated form, and since the active ingredient of the above pharmaceutical composition may be denatured or degraded by gastric acid, the oral composition may be administered into the mouth in a form coated with the active agent or formulated to protect it from degradation in the stomach, or in the form of an oral patch. Additionally, the above composition may be administered by any device capable of transporting the active substance to target cells. Effects of the invention

[0091] The micelle containing the novel peptide of the present invention can form a complex with a drug such as siRNA, and since the complex has excellent cell permeability and can effectively deliver the drug into the cell or tissue, the micelle can be utilized as a drug delivery vehicle. Brief explanation of the drawing

[0092] Figure 1 is a diagram showing the results of confirming whether the micelle-siRNA complex of the present invention is formed. Figure 2 is a diagram showing the results of confirming the stability level in serum of the micelle-siRNA complex of the present invention. Figure 3 is a diagram showing the results of evaluating the penetration level of the micellar-siRNA complex of the present invention into HaCat cells. Figure 4 is a diagram showing the results of evaluating the penetration level of the micellar-siRNA complex of the present invention into PC3 cells. Figure 5 is a figure showing the results of evaluating the penetration level of the micellar-siRNA complex of the present invention into KB cells. Figure 6 is a diagram showing the results of evaluating the target gene inhibitory efficacy of a micelle-siRNA complex containing siPARP1. Figure 7 is a diagram showing the results of evaluating the target gene inhibitory efficacy of a micelle-siRNA complex containing siFAK. Figure 8 is a figure showing the results of evaluating the target gene inhibitory efficacy of a micelle-siRNA complex containing siAR. Figure 9 is a figure showing the results of evaluating the inhibitory efficacy of two target genes of a micelle-siRNA complex containing siPARP1 and siFAK. Figure 10 is a figure showing the results of evaluating the inhibitory efficacy of two target proteins of a micelle-siRNA complex including siPARP1 and siFAK at an in vivo level. Figure 11 is a diagram showing the results of confirming the level of cytotoxicity following treatment with the micelle-siRNA complex of the present invention. Specific details for implementing the invention

[0093] The following examples will be explained in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0095] Preparation Example 1: Preparation of cell-permeable peptide

[0096] To prepare cell-permeable peptides, the following experiments were performed.

[0097] Specifically, as a hydrophilic peptide having cell-permeable characteristics, a peptide having the amino acid sequence 'KNRRKKKKQK' (Sequence No. 1) was prepared. In addition, a cell-permeable peptide was prepared in which histidine (H) was bound to the N-terminus and / or C-terminus of the above peptide.

[0098] The amino acid sequence of the cell-permeable peptide prepared above is listed in the table below.

[0099] No. Amino acid sequence (N → C) Sequence number 1 KNRRKKKKQK 1 2 KNRRKKKKQKHHHHHH 2 3 HHHKNRRKKKKQKHHH 3 4 HHHHHHKNRRKKKKQK 4

[0101] Preparation Example 2: Preparation of Amphiphilic Peptide

[0102] To prepare amphiphilic peptides, the following experiments were performed.

[0103] Specifically, to prepare an amphiphilic peptide, a peptide was prepared by attaching a hydrophobic substance to the N-terminus of the cell-permeable peptide prepared in Preparation Example 1, and as an example of the hydrophobic substance, stearic acid (represented as C18) was introduced. The amphiphilic peptide was prepared using a solid-phase synthesis (SPPS) method by commissioning WellPep Co., Ltd. (South Korea), and its specific structure is described in the table below.

[0104] name Peptide composition (N → C) Introduced hydrophobic material SCL1108 C18-KNRRKKKKQK C18 SCL1111 C18-KNRRKKKKQKHHHHHH C18 SCL1113 C18-HHHKNRRKKKKQKHHH C18 SCL1114 C18-HHHHHHKNRRKKKKQK C18

[0106] Preparation Example 3: Preparation of micelles containing an amphiphilic peptide

[0107] To prepare micelles containing the amphiphilic peptide prepared in Preparation Example 2 above, the following experiment was performed.

[0109] 3.1: Preparation of micelles containing amphiphilic peptides

[0110] To prepare micelles containing amphiphilic peptides, the following experiment was performed.

[0111] Specifically, the amphiphilic peptide prepared in Example 2 was dissolved in DMSO at a concentration of 50 mg / mL, and then added to 1 ml of PBS while stirring to prepare a final concentration of 1 mg / mL.

[0113] 3.2: Preparation of Lipid-Containing Micelles

[0114] To prepare micelles containing lipids, the following experiment was performed.

[0115] Specifically, as an example, to prepare a micelle containing 3% DSPE, the amphiphilic peptide combined with a hydrophobic substance prepared in Preparation Example 2 was dissolved in DMSO at a concentration of 50 mg / mL, and then 25 mg / mL of pegylated lipid (DSPE-PEG(2000)) was mixed as an example of a lipid to prepare a mixture such that the mass ratio of the peptide to DSPE is 97:3 (w / w) (the mass ratio can be adjusted according to the desired ratio), and the mixture was added to 1 ml of PBS while stirring to prepare a final concentration of 1 mg / mL.

[0116] Next, to prepare a micelle containing 3% DSPE and 2% FA as an example of a micelle containing a targeting ligand and lipids, we intended to additionally include FA (Folic acid) as an example of a targeting ligand. In the same manner as above, we prepared a mixture such that the peptide, DSPE, and FA were in a mass ratio of 95:3:2 (w / w / w) (the mass ratio can be adjusted according to the desired ratio), and added the mixture to 1 ml of PBS while stirring to prepare a final concentration of 1 mg / mL.

[0118] 3.3: Preparation of Micelles Containing Polymers

[0119] To prepare micelles containing polymers, the following experiment was performed.

[0120] Specifically, the amphiphilic peptide combined with a hydrophobic substance prepared in Preparation Example 2 above was dissolved in DMSO at a concentration of 50 mg / mL, and PLGA (polylactide-co-glycolide) was also dissolved in a separate DMSO at a concentration of 50 mg / mL as an example of a polymer. Next, to prepare micelles with a concentration of 1 mg / mL, the PLGA polymer and peptide were mixed so that the mass ratio was 7:3 (w / w) (the mass ratio can be adjusted according to the desired ratio), and a total of 20 μl of the mixture was added in 3 μl increments to 980 μL of DW while stirring at 800 rpm to achieve a final concentration of 1 mg / mL, and then stirring was maintained for an additional 5 minutes. Next, the above mixture was transferred to a tube and subjected to ultrasonic fragmentation (conditions of 30W, 5 sec-on / 5 sec-off pulse, total 2 min), and then subjected to an additional 5 minutes of ultrasonic fragmentation in a bathtub-type ultrasonic fragmenter to manufacture.

[0122] Preparation Example 4: Preparation of Micelle-SiRNA Complex

[0123] To prepare the micelle-siRNA complex, the following experiment was performed.

[0124] Specifically, the micelle prepared in Preparation Example 3 above was diluted in DEPC-DW, and the siRNA to be introduced into the complex was also diluted using DEPC-DW. Next, considering the mass ratio (w / w) of micelle to siRNA, the diluted micelle and siRNA were mixed in equal volumes (1:1), and then incubated at room temperature for 30 minutes to form a complex.

[0126] Example 1: Formation and Characterization of Amphiphilic Peptide-Based Micelles

[0127] To confirm whether amphiphilic peptide-based micelles were formed and their characteristics as prepared in Preparation Example 3 above, the following experiment was performed.

[0128] Specifically, the size of the prepared micelles was measured using photon correlation spectroscopy (PCS method) with a Zetasizer (Zetasizer Pro blue label, Malvern, US), and the average value was calculated by measuring the same sample at least 5 times. In addition, the zeta potential was measured using a Zetasizer (Zetasizer Pro blue label, Malvern, US), and the average value was calculated by measuring the same sample at least 5 times.

[0129] As a result of the above experiment, it was confirmed that micelles containing DSPE were effectively formed and exhibited the size and zeta potential listed in the table below.

[0130] SCM1111 SCM1113 SCM1114 SCM1108 DSPE(%) 20 10 20 10 20 10 20 10 size (nm) 10.7 11.6 8.4 7.1 12.7 12.5 10.8 9.7 zeta potential (mV) 3.8 8.2 7.2 16 12.2 16.7 3.3 2.4

[0132] Next, micelles containing various proportions of PLGA were also effectively formed and confirmed to exhibit the sizes and zeta potentials listed in the table below.

[0133] PLGA:CPP ratio (w / w) 5:5 6:4 7:3 8:2 9:1 size (nm) 91.2 ± 2.9 99.8 ± 3.0 112.1 ± 6.0 120.8 ± 6.7 138.2 ± 13.9 PDI 0.12 0.11 0.08 0.09 0.09 zeta potential(mV) 49.1 ± 0.2 45.0 ± 0.6 47.3 ± 0.3 47.7 ± 0.3 46.2 ± 0.6

[0135] Next, to further confirm the stability of micelles containing PLGA, micelles containing PLGA and peptide in a 7:3 ratio were stored for 10 days, and their characteristics were examined. It was confirmed that there was no difference before and after storage, indicating that the micelles were formed stably.

[0136] Storage period D+0 D+10 PLGA:CPP ratio 7:3 7:3 size (nm) 112.1 ± 6.0 110.3 ± 2.1 PDI 0.08 0.1 zeta potential (mV) 47.3 ± 0.3 47.1 ± 0.8

[0138] Example 2: Formation and Characterization of Micelle-SiRNA Complexes

[0139] To confirm whether the amphiphilic peptide-based micelle and siRNA complex prepared in Preparation Example 3 above was formed and to confirm its characteristics, the following experiment was performed.

[0141] 2.1: Confirmation of Complex Formation between Amphiphilic Peptide-Based Micelles and siRNA

[0142] In order to determine the optimal mixing ratio for the amphiphilic peptide-based micelles prepared in Preparation Example 3 to form a complex with siRNA, the degree of complex formation according to each ratio (micelle:siRNA) was examined. To this end, a gel retardation assay was performed, and the siRNA was stained with Midori Green Advance and visualized using a GelDoc go imaging system (BioRad, BR12009077). It was utilized that when siRNA forms a complex with micelles, the siRNA is not observed due to the interference effect of the peptide when stained with GelRed nucleic acid stain.

[0143] Specifically, as an example of an amphiphilic peptide-based micelle, a micelle containing SCM1108 peptide, a micelle containing SCM1108 peptide and 3% DSPE (SCM1108_3%DSPE), and a micelle containing SCM1108 peptide, 3% DSPE, and 2% FA (SCM1108_3%DSPE_2%FA) and siRNA were mixed to equal volumes (1:1) to make a 20 μL mixture while considering the mass ratio (w / w) of micelles:siRNA listed in the table below, and then incubated at room temperature for 30 minutes to form a complex. Next, 4 μl of 6x DNA loading dye was added to the mixture and mixed, then loaded onto a 2% agarose gel containing midori green advance and electrophoresis was performed at 50 V for 30 minutes, and the gel was visualized using a GelDoc Go imaging system (BioRad, BR12009077).

[0144] As a result of the above experiment, it was confirmed that a complex with siRNA is formed even in the case of micelles containing only amphiphilic peptides, and it was also confirmed that micelles containing lipids and / or ligands can also effectively form a complex with siRNA (Fig. 1).

[0146] 2.2: Determination of the size of micelles containing amphiphilic peptides and complexes containing siRNA

[0147] To confirm the size, etc., of the micelle containing the amphiphilic peptide and the complex containing siRNA prepared in Preparation Example 3 above, the following experiment was performed.

[0148] Specifically, as an example of an amphiphilic peptide-based micelle, a micelle containing the SCM1108 peptide was used, and the micelle was mixed with siRNA (PARP1, Thermo Fisher 4390824) to form a complex, and its size, etc., was measured through the method described in Example 1.

[0149] As a result, it was confirmed that the micelle-siRNA complex was effectively formed and exhibited the size and zeta potential listed in the table below.

[0150] micelle:siRNA ratio (w / w) 9:1 12:1 size (nm) 58.0 ± 50.9 56.7 ± 34.1 PDI 0.53 0.34 zeta potential (mV) 17.3 ± 7.3 10.5 ± 6.7

[0152] 2.3: Size determination of micelles containing amphiphilic peptides and lipids and complexes containing siRNA

[0153] In order to confirm the size, etc., of the micelle containing the amphiphilic peptide and lipid and the complex containing siRNA prepared in Preparation Example 3 above, the following experiment was performed.

[0154] Specifically, as an example of a micelle containing amphiphilic peptides and lipids, a micelle containing 3% DSPE was used, and the micelle and siRNA were mixed in a mass ratio of 9:1 to form a complex, and its size, etc., was measured using the method described in Example 1. As a result, it was confirmed that the micelle-siRNA complex was effectively formed and exhibited the size listed in the table below.

[0155] SCM1108_3%DSPE SCM1113_3%DSPE SCM1114_3%DSPE size (nm) 88.4±17.3 132.3±8.7 143±0.1

[0157] Next, micelles containing amphiphilic peptides and lipids were mixed with siRNA in various mass ratios to form complexes, and their size and other properties were measured using the method described in Example 1. As a result, it was confirmed that the micelle-siRNA complexes were effectively formed even in various mass ratios and exhibited the sizes listed in the table below.

[0158] micelle:siRNA ratio (w / w) 6:1 9:1 12:1 size (nm) 68.3 ± 4.1 88.4 ± 17.3 68.3 ± 28.1

[0160] Next, as an example of a micelle containing an amphiphilic peptide, lipids, and a ligand (FA), a micelle (SCM1108_3%DSPE_2%FA) containing SCM1108 peptide, 3% DSPE, and 2% FA was used. The micelle was mixed with one or more siRNAs (siPARP1 and siFAK) in a mass ratio of 12:1 to form a complex, and its size and other properties were measured using the method described in Example 1. As a result, it was confirmed that the micelle-siRNA complex was effectively formed and exhibited the size and zeta potential listed in the table below.

[0161] siRNA micelle only PARP1 FAK PARP1+FAK size (nm) 7.2 ± 40.4 127.2 ± 5.7 153.3 ± 1.1 108.7 ± 7.0 PDI 0.76 0.06 0.05 0.1 zeta potential (mV) 8.5 ± 0.8 -2.7 ± 1.4 -2.7 ± 1.4 -0.4 ± 1.0

[0163] 2.4: Size determination of micelles containing amphiphilic peptides and polymers, and complexes containing siRNA

[0164] To confirm the size, etc., of the micelle containing the amphiphilic peptide and polymer prepared in Preparation Example 3 above and the complex containing siRNA, the following experiment was performed.

[0165] Specifically, as an example of a micelle containing an amphiphilic peptide and a polymer, a micelle containing PLGA:peptide (SCM1108) in a 7:3 mass ratio was used, and the micelle and siRNA were mixed in various mass ratios to form a complex, and its size and other properties were measured using the method described in Example 1. As a result, it was confirmed that the micelle-siRNA complex was effectively formed and exhibited the size and zeta potential listed in the table below.

[0166] micelle:siRNA ratio (w / w) 5:1 10:1 15:1 20:1 size (nm) 114.2 ± 16.0 99.2 ± 14.1 101.2 ± 3.5 94.7 ± 17.6 PDI 0.42 0.33 0.17 0.36 zeta potential (mV) 8.3 ± 0.8 21.3 ± 1.2 30.6 ± 0.9 27.1 ± 0.7

[0168] Next, micelles containing amphiphilic peptides and polymers in various mass ratios were mixed with siRNA in a mass ratio of 10:1 to form a complex, and its size and other properties were measured using the method described in Example 1. As a result, it was confirmed that the micelle-siRNA complex was effectively formed and exhibited the size and zeta potential listed in the table below.

[0169] PLGA:CPP ratio 5:5 7:3 size (nm) 80.6 ± 2.0 95.4 ± 4.4 PDI 0.14 0.17 zeta potential(mV) 36.8 ± 1.9 36.6 ± 1.0

[0171] Example 3: Evaluation of Serum Stability of Micelle-SiRNA Complexes

[0172] To evaluate the serum stability of the micellar-siRNA complex prepared in Preparation Example 4 above, the following experiment was performed.

[0173] Specifically, as an example of an amphiphilic peptide-based micelle, a micelle containing the SCM1108 peptide prepared in Preparation Example 3, a micelle containing the SCM1108 peptide and 3% DSPE (SCM1108_3%DSPE), or a micelle containing the SCM1108 peptide, 3% DSPE, and 2% FA (SCM1108_3%DSPE_2%FA) was mixed with siRNA in a mass ratio of 12:1 to prepare a complex (9 μl), an equal volume of FBS (9 μl) to the complex was added, and the mixture was incubated at 37°C for 3, 6, 9, 12, 24, 48, or 72 hours. After the above incubation, 3 μg (2 μl) of heparin (1.5 μg / μl) was added to the sample and competitively reacted with the siRNA that had formed a complex with the micelle to separate the micelle and siRNA. The sample was then incubated at room temperature for 30 minutes. Afterward, 4 μl of 6x DNA loading dye was added to each sample and mixed. The samples were then loaded onto a 2% agarose gel containing midori green advance and subjected to electrophoresis at 50V for 30 minutes. The gel was visualized using a GelDoc Go imaging system (BioRad, BR12009077).

[0174] As a result of the above experiment, siRNA that was not bound to micelles degraded from 30 minutes onwards, and the remaining amount could not be confirmed. However, in the case of siRNA bound by forming a complex with the amphiphilic peptide-based micelle of the present invention, a remaining amount of up to 58% and a minimum of 31% was observed up to 48 hours (Fig. 2), indicating that the micelle-siRNA can significantly improve the stability of siRNA in serum.

[0176] Example 4: Evaluation of targeting efficacy of micellar-siRNA complexes loaded with targeting ligands

[0177] To evaluate the targeting efficacy of the micellar-siRNA complex prepared in Preparation Example 4 above following the loading of a targeting ligand, the following experiment was performed.

[0178] Specifically, folic acid (FA) was used as an example of a targeting ligand, and FA was added in various mass ratios to micelles containing the SCM1108 peptide prepared in Preparation Example 3 and 3% DSPE as an example of an amphiphilic peptide-based micelle. A complex was prepared by mixing the micelles with siRNA at a mass ratio of 12:1 or 9:1, and the siRNA terminals were labeled with FAM to confirm the cell permeability of the micelle-siRNA complex. Subsequently, the micelle-siRNA complex was treated with normal cells (HaCat), FA receptor-negative prostate cancer cells (PC3), or FA receptor-positive cervical cancer cells (KB), and the permeability to the cells was analyzed using a flow cytometry analyzer (FACS). For the above FACS analysis, each cell was treated with a micelle-siRNA complex and incubated for 24 hours, washed with PBS, and separated by trypsin treatment, and then measured using an attune NxT flow cytometer.

[0179] As a result of the above experiment, it was confirmed that the micelle-siRNA complex had a low penetration efficiency, with less than 2% penetration in normal HaCat cells and FA receptor-negative PC3 cells compared to the control group Lipofectamine, but showed a penetration rate equivalent to or greater than that of Lipofectamine in FA receptor-positive KB cells (Figs. 3 to 5 and Table 12), indicating that the micelle-siRNA complex of the present invention has significantly superior targeting efficiency due to the loaded targeting ligand.

[0180] cell line HaCaT PC3 KB P1 (%) Transmittance (%) P1 (%) Transmittance (%) P1 (%) Transmittance (%) control group 0.02 0.0 0.066 0.0 0.133 0.0 SiRNA 0.035 0.2 0.219 0.9 0.006 -0.2 Lipofectamine 8.546 100.0 16.666 100.0 8.038 100.0 SCM1108_3% DSPE 0.1% FA 0.117 1.1 0.162 0.6 8.066 100.4 1% FA 0.105 1.0 0.298 1.4 8.109 100.9 2% FA 0.173 1.8 0.084 0.1 7.642 95.0

[0182] Example 5: Evaluation of the target protein expression inhibition efficiency of the micellar-siRNA complex

[0183] To evaluate the efficacy of the micellar-siRNA complex comprising the micellar and siRNA of the present invention in inhibiting the expression levels of target genes and proteins translated therefrom, the following experiment was performed.

[0184] First, as an example of the micelle of the present invention, a complex was formed by mixing a micelle (SCM1108_3%DSPE_2%FA) containing SCM1108 peptide, 3% DSPE, and 2% FA with siRNA (siPARP1 or siFAK) in a mass ratio of 12:1. The prepared micelle-siRNA complex was transfected into KB cells, and after 2 hours, a medium containing 10% FBS was added and the cells were cultured for 48 hours. Subsequently, the cells were collected, and after RNA extraction and cDNA synthesis, the level of inhibition of the target gene by siRNA was determined using real-time qPCR (RT-qPCR). For the RT-qPCR, total RNA was purified according to the kit manual, and RNA was quantified using a nano-drop. 1 μg of RNA was taken, and reverse transcriptase and a poly-dT primer were added to synthesize cDNA. The corresponding mRNA was synthesized by adding PARP-1, FAK2, GAPDH primers (a loading control), a fluorescently detectable ROX dye, and transcription enzymes to the above-mentioned synthesized cDNA. Based on the ct values ​​measured by the program, the expression level of the corresponding mRNA relative to the control group was 2^-((ct gene -ct control gene )-(ct actin -ct control actin Calculated and compared according to the formula.

[0185] As a result of the above experiment, it was confirmed that the micelle-siRNA complex containing the micelle of the present invention exhibited a similar level of target gene inhibitory efficacy compared to the case using lipofectamine, which is a positive control (Fig. 6).

[0186] Next, as an example of the micelle of the present invention, a micelle (PLGA_SCM1108) containing SCM1108 peptide and the polymer PLGA was mixed with siRNA (siAR) in a mass ratio of 5:1 to form a complex, and after treating cells with the formed complex using the above method, the expression level of the target gene was evaluated.

[0187] As a result of the above experiment, it was confirmed that the micelle-siRNA complex containing the micelle of the present invention exhibited a similar level of target gene inhibitory efficacy compared to the case using lipofectamine, which is a positive control (Fig. 7).

[0188] Based on the above results, it can be seen that the micelles of the present invention can effectively deliver siRNA to suppress target genes.

[0190] Example 6: Evaluation of the Inhibitory Efficiency of Two Target Protein Expressions by Micelle-SiRNA Complexes

[0191] To evaluate whether the micell-siRNA complex of the present invention, comprising micellar and two types of siRNA, can effectively suppress the expression levels of two types of genes targeted by each and proteins translated therefrom, the following experiment was performed.

[0193] 6.1: Evaluation at the in vitro level

[0194] As an example of the micelle of the present invention, a complex was formed by mixing a micelle (SCM1108_3%DSPE_2%FA) containing SCM1108 peptide, 3% DSPE, and 2% FA with two types of siRNA (siPARP1 and siFAK) in a mass ratio of 12:1. In addition, the two types of siRNA were mixed to form a complex such that the mass ratio of siPARP1 and siFAK was 50:25, 50:20, or 50:10 (w / w). The prepared micelle-siRNA complex was transfected into KB cells, and the expression level of the target gene was evaluated using the method described in Example 5.

[0195] As a result of the above experiment, it was confirmed that even in the case of micelle complexes using two types of siRNA, each siRNA can act on a target gene to suppress expression, and it was confirmed that the inhibitory efficacy of each target gene can be maintained even when the two types of siRNA are mixed in various mass ratios (Fig. 8).

[0197] 6.2: Evaluation at the in vivo level

[0198] For xenograft, 5x10 cultured KB cells 5 The mice were injected into the right shoulder of the mice to form a cell / mouse, and adverse reactions were observed while rearing for 3 to 4 weeks to allow tumor formation, and the volume of the tumor formed before the injection of the micelle-siRNA complex was measured.

[0199] In addition, as an example of the micelle of the present invention, a complex was formed by mixing a micelle (SCM1108_3%DSPE_2%FA) containing SCM1108 peptide, 3% DSPE, and 2% FA with two types of siRNA (siPARP1 and siFAK) in a mass ratio of 12:1. In addition, the two types of siRNA, siPARP1 and siFAK, were mixed in a mass ratio of 5:1. The total injection amount of siRNA was prepared as 2.5 μg / mouse for 1x and 12.5 μg / mouse, which is five times the amount, for 5x. The above-prepared complex was injected into mice via the intra-tumor (IT) method at a dose of 1.6 mg / kg, and the mice were sacrificed after 3 days to extract xenograft tumor tissue. The tissue extracted from one mouse was divided into pieces of approximately 25 mg to obtain a total of 3 pieces, and one piece was divided in half to extract RNA for RT-qPCR or protein for Western blotting.

[0200] First, the expression level of the target gene was evaluated using RT-qPCR with the method described in Example 5, and it was confirmed that the two types of siRNA included in the complex could effectively suppress the expression of each target gene (Fig. 9).

[0201] Next, the expression levels of proteins translated from target genes were determined using Western blotting, and it was confirmed that the expression levels of the proteins targeted by the two types of siRNAs included in the complex were inhibited to 88% and 91%, respectively (Fig. 10).

[0202] Based on the above results, it can be seen that when the micelle of the present invention forms a complex with two or more types of siRNA, it can be delivered into a cell or tissue, and each siRNA can effectively inhibit a target gene.

[0204] Example 7: Evaluation of cell targeting efficacy of micellar-siRNA complexes

[0205] To evaluate the targeting efficacy of the micelle-siRNA complex containing the targeting substance prepared in Preparation Example 4 above, the following experiment was performed.

[0206] Specifically, a complex was prepared by mixing a micelle (SCM1108_3%DSPE_2%FA) containing the SCM1108 peptide prepared in Preparation Example 3 and 3% DSPE as an example of a micelle containing amphiphilic peptides and lipids, and 2% FA as an example of a targeting substance, with two types of siRNA (siPARP1:siFAK = 5:1 mass ratio) in a mass ratio of 12:1, and this complex was treated on three types of cancer cell lines (KB, HeLa, and HaCaT). Meanwhile, the gynecological cancer cell lines KB and HeLa are known to have a high distribution of folate receptors, with the distribution of folate receptors in KB cells being more than twice as high as in HeLa cells, while HaCaT cells are known to have a low distribution of folate receptors. The above complex was serially diluted twofold from 100 μg / mL to 0.4 μg / mL to produce nine different concentrations, which were treated for 24 hours, followed by WST-1 (water-soluble tetrazolium salt-1, EZ-assay kit, Doogenbio) analysis. For WST-1 analysis, 0.1 ml of WST-1 solution was added to wells of cells that had completed 24 hours of culture and incubated for 3 hours; subsequently, absorbance was measured at 450 nm using a microplate spectrophotometer (SpectraMax iD3, Molecular Devices). Dose-dependent graphs were obtained using the Softmax program, and logistic 4-parameter regression was performed to determine the CC 50 It produced.

[0207] As a result of the above experiment, it can be seen that siPARP1 and siFAK delivered into the cell via the above carrier exhibit toxicity against cancer cells, and CC 50It was confirmed that the levels were 15 μg / mL in KB cells, 45.8 μg / mL in HeLa cells, and 81.6 μg / mL in HaCaT cells (Fig. 11 and Table 13).

[0208] sample KB cell HeLa cell HaCaT cell Conc. (ug / mL) viability avg. (%) cv% avg. (%) cv% avg. (%) cv% 100 10.2 5.6 1.9 9.1 10.9 33.8 50 11.1 4.3 43.7 10.1 83.7 4.8 25 28 9.1 94.6 4.2 98.3 3.6 12.5 66.3 7.4 118.6 4.1 104.2 1.2 6.3 93.3 1.1 115.3 3.4 98.8 1.8 3.1 92.4 0.4 110.4 6.9 105.1 2.8 1.6 105 0.7 105.4 5.4 95.5 0.7 0.8 105.9 1.7 99.2 5.8 99.6 2.2 0.4 103.3 2 101.2 3 98.0 1.5 CC 50 14.9 μg / mL 45.8 μg / mL 81.6 μg / mL

[0210] Based on the above results, in KB cells, where the distribution ratio of folate receptors present in the cell is relatively high, the siRNA of the micellar-siRNA complex is effectively delivered, and CC 50 In HaCaT, where the distribution ratio was low and the delivery efficiency of the micelle-siRNA complex was low, the high CC was evaluated as low. 50 As shown, it can be seen that the cancer cell toxicity caused by the siRNA is a result of the targeting efficacy of the complex via receptor-mediated endocytosis based on the targeting substance included in the complex.

[0212] 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.

Claims

Claim 1 A peptide composed of the amino acid sequence of SEQ ID NO.

1. Claim 2 A peptide composed of the amino acid sequence of SEQ ID NO. 1 and a peptide comprising histidine (H) at one or more of the amino terminus and carboxyl terminus of said peptide. Claim 3 A peptide according to claim 2, wherein the peptide further comprises 1 to 20 histidines. Claim 4 A peptide according to claim 2, wherein the peptide further comprises a polyhistidine peptide composed of one histidine or two to ten histidines at one or more of an amino terminus and a carboxyl terminus. Claim 5 A peptide according to claim 2, wherein the peptide comprises one or more sequences selected from the group consisting of SEQ ID NOs 2 to 4. Claim 6 A micelle comprising the peptide of any one of claims 1 to 5. Claim 7 In claim 6, the micelle is a cell-permeable mouselle. Claim 8 The micelle of claim 7, wherein the micelle is formed by the self-assembly of the peptide. Claim 9 A micelle according to claim 6, wherein the micelle further comprises a hydrophobic substance at the amino terminus or carboxyl terminus of the peptide, the hydrophobic substance comprising one or more hydrophobic moietyes selected from the group consisting of sterols and fatty acids. Claim 10 delete Claim 11 A micelle according to claim 6, wherein the micelle further comprises one or more of a targeting substance, a labeling substance, and a linker for linking the targeting substance or the labeling substance. Claim 12 In claim 6, the micelle is a micelle further comprising one or more of lipids and biocompatible polymers, wherein the lipid is DSPE (distearoylphosphatidylethanolamine), DSPE-PEG, DSPC (distearoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), POPC (palmitoyloleoylphosphatidylcholine;The biocompatible polymer comprises one or more selected from the group consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), EPC (egg phosphatidylcholine), DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), PE (phosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]), sphingomyelin, and DMG-PEG (1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol). A micelle comprising one or more selected from the group consisting of PLGA (polylactide-co-glycolide), PLA (polylactic acid), PLLA (poly-L-lactic acid), PDLLA (poly-d,l-lactic acid), and PDLA (poly-D-lactic acid). Claim 13 delete Claim 14 delete Claim 15 A drug delivery complex comprising a micelle of claim 6; and a desired drug, wherein the drug comprises a nucleic acid. Claim 16 delete Claim 17 A drug delivery complex according to claim 15, wherein the drug comprises one or more selected from the group consisting of DNA, mRNA, siRNA, microRNA, ASO, gapmer, aptamer, antagomir, agomir, and oligonucleotide. Claim 18 A drug delivery composition comprising the micelle of claim 6. Claim 19 delete

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