Collagen-binding membrane-permeable peptide, and carrier that contains said peptide and collagen or collagen derivative

Abstract

The present invention addresses the problem of providing a carrier for easily introducing a medicine or the like into cells.　It was found that the problem can be solved by: a collagen-binding membrane-permeable peptide comprising a collagen-binding domain, a membrane fusion domain, and a membrane-permeable domain; and a carrier comprising said peptide and collagen or a collagen derivative. Due to the foregoing, the present disclosure was completed.

Description

Collagen-binding membrane-permeable peptide and carrier containing said peptide and collagen or collagen derivative

[0001] The present disclosure relates to a collagen-binding membrane-permeable peptide for introducing a desired protein, peptide, nucleic acid, low-molecular-weight compound, etc. into target cells, a carrier comprising the peptide and collagen or a collagen derivative, a cell culture device, a medical device, a drug, and a method for changing the properties of target cells using the carrier. This application claims priority to Japanese Patent Application No. 2023-121675, which is incorporated herein by reference.

[0002] (Collagen-based carriers) Collagen is known as a sustained-release carrier for drugs (see Patent Documents 1 and 2, Non-Patent Document 1), and furthermore, kits for introducing nucleic acids into target cells are commercially available (see Non-Patent Document 2).

[0003] (Patent Document 1) Patent Document 1 discloses a "cell transfection array for nucleic acid introduction comprising atelocollagen, a nucleic acid introduction agent, and a nucleic acid."

[0004] (Patent Document 2) Patent Document 2 discloses "complex particles containing atelocollagen and a desired nucleic acid, wherein the nucleic acid is single-stranded or double-stranded and the major axis of the particle is 100 μm or less." The configuration of the complex particles disclosed in this patent document differs from the configuration of the carrier of the present disclosure.

[0005] (Patent Document 3) Patent Document 3 discloses "a carrier comprising collagen or a collagen derivative to which a cell membrane-permeable peptide has been added." The configuration of the collagen to which a cell membrane-permeable peptide has been added disclosed in this patent document is different from the configuration of the collagen-binding membrane-permeable peptide disclosed in the present disclosure. Specifically, the cell membrane-permeable peptide disclosed in this patent document is covalently bonded to the carrier.

[0006] Patent 2006-6262, Patent 4081436, Patent 2014-210730

[0007] Cancer Res. 2004 May15;64(10):3365-70http: / / www.atelocollagen.com / atelogene / japanese / index.html /

[0008] Lipid nanoparticles, liposomes, and aptamers have been reported as DDS for nucleic acid drugs. However, there are various issues, such as the need for expensive equipment to modify nucleic acid drugs and the risk of allergic reactions due to PEG modification. Peptide-based DDS are also being researched, but because they are designed to be covalently linked to nucleic acid drugs or carrier proteins, they must be synthesized for each target substance. To address the above issues, the goal of this project is to provide a carrier that can easily deliver drugs and other substances into cells.

[0009] As a result of research to solve the above problems, the present inventors have found that a collagen-binding membrane-permeable peptide containing a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain, as well as a carrier containing said peptide and collagen or a collagen derivative, can solve the above problems, and have thus completed the present disclosure.

[0010] The present disclosure is as follows: 1. A collagen-binding transmembrane peptide comprising a collagen-binding domain, a membrane fusion domain, and a membrane transmembrane domain. 2. The collagen-binding transmembrane peptide according to the preceding paragraph 1, in which a linker is inserted between each domain. 3. The collagen-binding domain is TKKTLRT (SEQ ID NO: 1), and the membrane fusion domain is X1FVIX5AX7VLX. 10 ALX 13 X 14 X 15 IX 17 X 18 I (SEQ ID NO: 2) or PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36), wherein X1 is P or A, X5 is G or A, X7 is G or A, 10 is G or A, X 13 is G or A, X 14 is T or A, X 15 is G or A, X 17 is G or A, and X 18is G or A, and the transmembrane domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), RQIKIWFQNRRMKWKK (SEQ ID NO: 5), GRKKRRQRRRPQ (SEQ ID NO: 6), rrrrrrrr (SEQ ID NO: 7), AFLGWLGAWGTMGWSPKKKRK (SEQ ID NO: 8), RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9), RRLSYSRRRF (SEQ ID NO: 10), PIRRRKKLRRLK (SEQ ID NO: 11), RRQRRTSKLMKR (SEQ ID NO: 12), RRRRNRTRRNRRRVR (SEQ ID NO: 13), KMTRAQRRAAARRNRWTAR (SEQ ID NO: 14), TRRQRTRRARRNR (SEQ ID NO: 15), GRKKRRQRRRPPQ (SEQ ID NO: 16), GRRRRRRRRRPPQ (SEQ ID NO: 17), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 18), KLALKLALKLALALKLA (SEQ ID NO: 19), MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 20), or GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 21). 3. The collagen-binding membrane-permeable peptide according to item 1 or 2 above, 4. The collagen-binding membrane-permeable peptide according to item 3 above, wherein the collagen-binding domain is TKKTLRT (SEQ ID NO: 1), the membrane fusion domain is PFVIGAGVLGALGTGIGGI (SEQ ID NO: 22), and the membrane-transmitting domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), or RQIKIWFQNRRMKWKK (SEQ ID NO: 5). 5. The collagen-binding membrane-permeable peptide according to item 3 above, wherein the collagen-binding domain is TKKTLRT (SEQ ID NO: 1), the membrane fusion domain is PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36), and the membrane-transmitting domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), or RQIKIWFQNRRMKWKK (SEQ ID NO: 5). 6. A carrier comprising the collagen-binding membrane-permeable peptide according to item 4 above and collagen or a collagen derivative. 7. A carrier comprising the collagen-binding membrane-permeable peptide according to item 5 above and collagen or a collagen derivative.8. The carrier according to the preceding paragraph 6, further comprising a target for delivery. 9. The carrier according to the preceding paragraph 7, further comprising a target for delivery. 10. The carrier according to the preceding paragraph 8, wherein the target for delivery is a protein, peptide, nucleic acid, and / or low molecular weight compound. 11. The carrier according to the preceding paragraph 8, wherein the collagen-binding membrane-permeable peptide, the collagen or collagen derivative, and / or the target for delivery form a complex. 12. A medical device having the carrier according to the preceding paragraph 8 applied to its surface. 13. A cell culture instrument having the carrier according to the preceding paragraph 8 applied to its cell culture surface. 14. A drug comprising the carrier according to the preceding paragraph 8. 15. 15. A delivery vehicle for cancer treatment, comprising a collagen-binding membrane-permeable peptide comprising a collagen-binding domain, a membrane fusion domain, and a membrane-transmitting domain, collagen or a collagen derivative, and a cancer therapeutic agent as a delivery target, wherein the collagen-binding domain is TKKTLRT (SEQ ID NO: 1), the membrane fusion domain is PFVIGAGVLGALGTGIGGI (SEQ ID NO: 22) or PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36), and the membrane-transmitting domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), or RQIKIWFQNRRMKWKK (SEQ ID NO: 5). A method for administering a target cell with a target substance, the method comprising the steps of: administering to a mammal, including a human, a carrier comprising a collagen-binding membrane-permeable peptide, collagen or a collagen derivative, and the target substance, the carrier comprising a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain, wherein the collagen-binding domain is TKKTLRT (SEQ ID NO: 1), and the membrane fusion domain is X1FVIX5AX7VLX. 10 ALX 13 X 14 X 15 IX 17 X 18 I (SEQ ID NO: 2) or PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36), wherein X1 is P or A, X5 is G or A, X7 is G or A, 10 is G or A, X 13 is G or A, X 14 is T or A, X 15 is G or A, X17 is G or A, and X 18 is G or A, and the transmembrane domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), RQIKIWFQNRRMKWKK (SEQ ID NO: 5), GRKKRRQRRRPQ (SEQ ID NO: 6), rrrrrrrr 16. A method for administering a delivery target to a target cell, wherein the delivery target is selected from the group consisting of (SEQ ID NO: 7), AFLGWLGAWGTMGWSPKKKRK (SEQ ID NO: 8), RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9), RRLSYSRRRF (SEQ ID NO: 10), PIRRRKKLRRLK (SEQ ID NO: 11), RRQRRTSKLMKR (SEQ ID NO: 12), RRRRNRTRRNRRRVR (SEQ ID NO: 13), KMTRAQRRAAARRNRWTAR (SEQ ID NO: 14), TRRQRTRRARRNR (SEQ ID NO: 15), GRKKRRQRRRPPQ (SEQ ID NO: 16), GRRRRRRRRRPPQ (SEQ ID NO: 17), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 18), KLALKLALKLALALKLA (SEQ ID NO: 19), MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 20), and GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 21). 17. The method of administering according to the preceding item 16, wherein the object to be delivered is a cancer therapeutic agent and the target cells are cancer cells. 18. Use of a collagen-binding membrane-permeable peptide comprising a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain in the manufacture of a carrier, wherein the collagen-binding domain is TKKTLRT (SEQ ID NO: 1), and the membrane fusion domain is X1FVIX5AX7VLX 10 ALX 13 X 14 X 15 IX 17 X 18 I (SEQ ID NO: 2) or PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36), wherein X1 is P or A, X5 is G or A, X7 is G or A, 10 is G or A, X 13 is G or A, X 14 is T or A, X 15 is G or A, X 17 is G or A, and X18 is G or A, and the transmembrane domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), RQIKIWFQNRRMKWKK (SEQ ID NO: 5), GRKKRRQRRRPQ (SEQ ID NO: 6), rrrrrrrr (SEQ ID NO: 7), AFLGWLGAWGTMGWSPKKKRK (SEQ ID NO: 8), RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9), RRLSYSRRRF (SEQ ID NO: 10), PIRRRKKLRRLK (SEQ ID NO: 11), RRQRRTSKLMKR (SEQ ID NO: 12), RRRRNRTRRNRRRVR (SEQ ID NO: 13), KMTRAQRRAAARRNRWTAR (SEQ ID NO: 14), TRRQRTRRARRNR (SEQ ID NO: 15), GRKKRRQRRRPPQ (SEQ ID NO: 16), GRRRRRRRRRPPQ (SEQ ID NO: 17), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 18), KLALKLALKLALALKLA (SEQ ID NO: 19), MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 20) or GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 21).

[0011] The collagen-binding membrane-permeable peptide and a carrier comprising the peptide and collagen or a collagen derivative of the present disclosure have one or more of the following effects: (1) They have a high efficiency of introducing the target into target cells. (2) They form a complex (particularly a ternary complex) simply by mixing the collagen-binding membrane-permeable peptide, collagen or a collagen derivative, and the target. (3) By introducing a linker (particularly a His linker) between each domain of the collagen-binding membrane-permeable peptide, the types of membrane fusion domain and membrane-permeable domain can be easily replaced. (4) They can be introduced with high efficiency even into target cells into which the target is difficult to introduce.

[0012] Synthesis scheme for the collagen-binding membrane-permeable peptide of the present disclosure. Confirmation of synthesis of CBD-S19-TAT of the present disclosure. Confirmation of synthesis of CBD-S19-Penetratin of the present disclosure. Schematic diagram of a pull-down assay using a Ni affinity column. Confirmation of complex formation between a carrier (atelocollagen (AC) and a collagen-binding membrane-permeable peptide) of the present disclosure. Confirmation of complex formation between AC and a delivery target (siRNA). Evaluation of the cytotoxicity of a carrier containing a delivery target of the present disclosure. Evaluation of RNA interference (RNAi) by a carrier of the present disclosure. Confirmation of the RNAi effect of a carrier containing CBD-S19-TAT of the present disclosure. Confirmation of the RNAi effect of a carrier containing CBD-S19-Penetratin of the present disclosure. Confirmation of the uptake of the delivery target contained in a carrier of the present disclosure into each cell.

[0013] (Subject of the present disclosure) The subject of the present disclosure is a "collagen-binding membrane-permeable peptide (hereinafter, may be referred to as "the peptide of the present disclosure")," a "carrier containing the peptide and collagen or a collagen derivative (hereinafter, may be referred to as "the carrier of the present disclosure")," a "cell culture instrument in which the carrier is applied to a cell culture surface (hereinafter, may be referred to as "the cell culture instrument of the present disclosure")," and a "drug containing the carrier (hereinafter, may be referred to as "the drug of the present disclosure")."

[0014] The "carrier" of the present disclosure has the function of delivering a target substance to a cell {a target cell (a cell into which the target substance is introduced)}. Furthermore, the carrier of the present disclosure not only delivers a target substance to a cell, but also has one or more of a sustained release function, a long-term retention function in the target cell, a specific localization function around the target cell, and a protection function of the target substance from degradation in the body.

[0015] (Peptides of the Present Disclosure) The peptides of the present disclosure contain at least a collagen-binding domain, a membrane-fusion domain, and a transmembrane domain. Furthermore, if necessary, a linker known per se (particularly a His linker (consisting of 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive His)) may be introduced between each domain. Additionally, the order of each domain in the peptide (from N-terminus to C-terminus) is not particularly limited, and examples include: collagen-binding domain-membrane-fusion domain-transmembrane domain collagen-binding domain-transmembrane domain-membrane fusion domain-collagen-binding domain-transmembrane domain membrane-fusion domain-transmembrane domain-collagen-binding domain membrane-fusion domain-transmembrane domain-collagen-binding domain membrane-fusion domain transmembrane domain-membrane fusion domain-collagen-binding domain The peptides of the present disclosure also include protected derivatives, glycosylated derivatives, acylated derivatives, or acetylated derivatives of peptides containing each of the domains shown below. The peptides of the present disclosure also include peptides that share 90 to 99% (95 to 99%, 97 to 99%, 98 to 99%) or more identity with peptides containing each of the domains shown below and have substantially the same activity as the peptides. Furthermore, the peptides of the present disclosure also include peptides that have 1 to 5 (1, 2, 3, or 4) amino acid substitutions, deletions, insertions, and / or additions in peptides containing each of the domains shown below and have substantially the same activity as the peptides. From the viewpoint of not altering the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptides (amino acids) when introducing mutations or substitutions into peptides (amino acids), for example, mutual substitutions between homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) are easily conceivable.

[0016] (Collagen-binding domain) The collagen-binding domain is not particularly limited as long as it has a sequence that allows the peptide of the present disclosure to bind to collagen. For example, TKKTLRT (SEQ ID NO: 1) can be exemplified.

[0017] (Membrane fusion domain) The membrane fusion domain is not particularly limited as long as it has a sequence that allows the peptide of the present disclosure to fuse with a cell membrane. For example, the following sequence can be exemplified: X1FVIX5AX7VLX 10 ALX 13 X 14 X 15 IX 17 X 18 I (SEQ ID NO: 2) PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36) X1 is P or A, X5 is G or A, X7 is G or A, X 10 is G or A, X 13 is G or A, X 14 is T or A, X 15 is G or A, X 17 is G or A, and X 18 is G or A. More specifically, PFVIGAGVLGALGTGIGGI (SEQ ID NO: 22) AFVIGAGVLGALGTGIGGI (SEQ ID NO: 26) PFVIAAGVLGALGTGIGGI (SEQ ID NO: 27) PFVIGAAVLGALGTGIGGI (SEQ ID NO: 28) PFVIGAGVLAALGTGIGGI (SEQ ID NO: 29) PFVIGAGVLGALATGIGGI (SEQ ID NO: 30) PFVIGAGVLGALGAGIGGI (SEQ ID NO: 31) PFVIGAGVLGALGTAIGGI (SEQ ID NO: 32) PFVIGAGVLGALGTGIAGI (SEQ ID NO: 33) PFVIGAGVLGALGTGIGAI (SEQ ID NO: 34) PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36) The membrane fusion domains, which are the peptides set forth in SEQ ID NOs: 26 to 34 and SEQ ID NO: 36, have the same effect as the membrane fusion domain, which is the peptide set forth in SEQ ID NO: 22 (see Biochemical and Biophysical Research Communications 586 (2022) 63-67, Journal of Biological Chemistry 298(7) (2022) 102097).

[0018] (Membrane-transmitting domain) The membrane-transmitting domain is not particularly limited as long as it has a sequence that allows the peptide of the present disclosure to penetrate a cell membrane. For example, the following known sequences can be exemplified: YGRKKRRQRRR (SEQ ID NO: 3) RKKRRQRRR (SEQ ID NO: 4) RQIKIWFQNRRMKWKK (SEQ ID NO: 5) GRKKRRQRRRPQ (SEQ ID NO: 6) rrrrrrrr (SEQ ID NO: 7) AFLGWLGAWGTMGWSPKKKRK (SEQ ID NO: 8) RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9) RRLSYSRRRF (SEQ ID NO: 10) PIRRRKKLRRLK (SEQ ID NO: 11) RRQRRTSKLMKR (SEQ ID NO: 12) RRRRNRTRRNRRRVR (SEQ ID NO: 13) KMTRAQRRAAARRNRWTAR (SEQ ID NO: 14) TRRQRTRRARRNR (SEQ ID NO: 15) GRKKRRQRRRPPQ (SEQ ID NO: 16) GRRRRRRRRRPPQ (SEQ ID NO: 17) GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 18) KLALKLALKLALALKLA (SEQ ID NO: 19) MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 20) GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 21)

[0019] (Collagen or Collagen Derivative) In the present disclosure, "collagen or collagen derivative" refers to any "collagen or collagen derivative" typically used in the medical, cosmetic, industrial, and food fields. It is preferable to use soluble or solubilized collagen. Soluble collagen is soluble in acidic or neutral water or salt solutions. Solubilized collagen includes enzyme-solubilized collagen solubilized with an enzyme and alkali-solubilized collagen solubilized with an alkali, both of which are preferably capable of passing through a membrane filter with a pore size of 1 micrometer. Collagen extracted from any animal species can be used, but is preferably extracted from vertebrates, more preferably from mammals, birds, or fish, and even more preferably from mammals or birds with high denaturation temperatures. Any type of collagen may be used, but types I to V are preferred due to their abundance in animal bodies. Specific examples include type I collagen extracted with acid from mammalian dermis, and more preferably type I collagen extracted with acid from calf dermis and type I collagen produced by genetic engineering. From the standpoint of safety, atelocollagen from which highly antigenic telopeptides have been enzymatically removed or atelocollagen produced by genetic engineering is desirable, and atelocollagen having 3 or fewer tyrosine residues per 1,000 residues is more preferred. The preferred collagen or collagen derivative of the present disclosure is atelocollagen.

[0020] (Method for Producing a Carrier of the Present Disclosure) The method for producing a carrier of the present disclosure can be carried out by mixing the peptide of the present disclosure, the collagen or collagen derivative of the present disclosure, and the target to be transported by a method known per se. This forms a complex (particularly a ternary complex), which has the following effects: (1) It plays a role in protecting the nucleic acid carrier from nucleases in vivo. (2) By binding the peptide of the present disclosure to the collagen / carrier (e.g., nucleic acid) of the present disclosure, the carrier can be efficiently introduced into cells (cytoplasm). The collagen-binding membrane-permeable peptide of the present disclosure is preferably not covalently bound in the carrier.

[0021] (Composition of the Carrier of the Present Disclosure) The carrier of the present disclosure may contain, in addition to the peptide of the present disclosure, the collagen or collagen derivative of the present disclosure, and the carrier, biocompatible materials, additives, etc. Examples of biocompatible materials include gelatin, fibrin, albumin, hyaluronic acid, heparin, chondroitin sulfate, chitin, chitosan, alginic acid, pectin, agarose, hydroxyapatite, polypropylene, polyethylene, polydimethylsiloxane, glycolic acid, lactic acid, or amino acid polymers or copolymers thereof, or mixtures of two or more of these biocompatible materials. Examples of additives include isotonicity agents, pH adjusters, and soothing agents when used as injections, and excipients, disintegrants, and coating agents when used as solid preparations. Specific examples include salts and sugars used to maintain a pH of 6-8 or to maintain isotonicity with cells. Furthermore, the carrier of the present disclosure may be in the form of a solid or a solution. When the carrier of the present disclosure is in a solid form, it is loaded onto the desired cells as is or in a solution form using purified water, physiological saline, a buffer solution isotonic with the body, or the like.

[0022] (Uses of the carrier of the present disclosure) The uses of the carrier of the present disclosure are not particularly limited, but can be used for drugs (particularly, cancer treatment agents), medical devices, cell culture devices, cosmetic ingredients, fertilizers, food additives, labeling agents, etc.

[0023] (Method of administering the carrier of the present disclosure) The carrier of the present disclosure can be administered to a living body (an animal including a human, particularly a mammal including a human) by any of oral administration, injection, eye drop, nasal drop, pulmonary administration, and absorption through the skin, preferably by injection. The administration site can be selected depending on the disease, but it can also be placed directly at the required site (e.g., cancer cells, cancer tissue) during surgery. For example, the carrier of the present disclosure can be administered systemically by intravenous injection (infusion, etc.), or locally by injection into the affected area (cancer cells, cancer tissue, etc.).

[0024] (Transportation Target) The "transportation target" in the present disclosure is not particularly limited, and examples thereof include proteins, peptides, nucleic acids, and low-molecular-weight compounds.

[0025] (Proteins) Examples include, but are not limited to, enzymes, agonists / antagonists of target receptors, receptors themselves, antibodies, etc. (Peptides) Examples include, but are not limited to, low-molecular-weight proteins with enzymatic activity, synthesized portions of functional proteins, and agonists / antagonists of target receptors. (Low-molecular-weight compounds) Examples include, but are not limited to, low-molecular-weight pharmaceuticals such as anticancer drugs that are specifically effective in killing tumor cells, and compounds that enhance or suppress the physiological activity of cells. (Nucleic acids) The "nucleic acid to be delivered" in this disclosure may be a polynucleotide or an oligonucleotide, or may be a DNA or RNA molecule. In the case of a DNA molecule, it may be a plasmid DNA, cDNA, genomic DNA, or synthetic DNA. Furthermore, both DNA and RNA may be double-stranded or single-stranded. In the case of a single-stranded molecule, it may be the coding strand or non-coding strand. "Nucleic acid" includes DNA derivatives and RNA derivatives, and these derivatives refer to nucleic acids having phosphorothioate bonds or nucleic acids in which the phosphate, sugar, or base moieties of internucleotides have been chemically modified to prevent enzymatic degradation. "Nucleic acid" also includes viruses such as adenoviruses and retroviruses. When the nucleic acid is a vector used in gene therapy, such as plasmid DNA or a virus, it is preferably in a form that is configured to express the encoded genetic information within a cell when introduced into the cell, such as a vector containing elements necessary for expression of the target gene, such as a promoter, or elements that enable integration into a chromosome.

[0026] (Medical Device or Cell Culture Device) The present disclosure also covers medical devices or cell culture devices having a carrier of the present disclosure coated on the surface. Coating a carrier of the present disclosure onto a solid surface and contacting target cells therewith improves the efficiency of delivery of the target compared to adding a carrier of the present disclosure dropwise onto the target cells. Medical devices of the present disclosure include artificial organs, more specifically, artificial blood vessels, medical device stents for reinforcing blood vessels, adhesive sheets, or artificial hearts. Cell culture devices of the present disclosure include petri dishes, flasks, 96-well microplates, three-dimensional culture supports, and the like, which are commonly used in cell culture experiments.

[0027] (Method for Investigating the Function of a Gene or Protein in a Target Cell) Using the carrier of the present disclosure, the function of a gene or protein in a target cell can be easily investigated. For example, a method for investigating the function of a gene by introducing a plasmid DNA incorporating the gene into the cell and expressing the gene, or a method for investigating the function of a gene by introducing an siRNA nucleic acid that suppresses the expression of the gene into the cell and suppressing the expression of the gene, are useful. A specific measurement method involves mixing a plasmid DNA expressing a gene of interest, an adenovirus vector, or an siRNA nucleic acid that suppresses the expression of the gene of interest with the carrier of the present disclosure, and applying and arranging the mixture on a solid phase of a culture plate. After drying and immobilizing the applied carrier on the solid phase, cells are seeded and cultured on the plate for several days. The applied carrier is efficiently introduced into the cells that adhere to the applied portion, expressing or suppressing the expression of the gene of interest for a long period of time. After several days, the function of the targeted gene can be elucidated by examining the cell proliferation rate, morphology (phenotype), the state of gene expression within the cell (gene expression level), or the type and amount of protein produced by the cell.

[0028] (Method for screening delivery targets capable of treating diseases) Using the delivery bodies of the present disclosure, it is possible to screen for candidate substances that can treat various diseases, such as genetic diseases, cancer, AIDS, rheumatoid arthritis, and lifestyle-related diseases. For example, a candidate substance (e.g., a nucleic acid) whose therapeutic effect on a disease is to be investigated is mixed with the delivery body of the present disclosure on a solid phase, and the mixture is applied and aligned on the solid phase of a culture plate. After the applied delivery body is dried and fixed on the solid phase, cells are seeded and cultured on the plate for several days. The effect of the nucleic acid can be analyzed by changes in cell phenotype, cell death, cell proliferation, intracellular gene expression patterns, and the type and amount of protein produced.

[0029] (Method for changing the properties of target cells using a carrier) The properties of target cells can be changed by using the carrier of the present disclosure. For example, if an antagonist that suppresses the activity of a specific intracellular receptor is administered to a target cell as a carrier, the target cell can have lower receptor activity (the properties of the target cell can be changed) compared to target cells that have not been administered with the antagonist. In addition, if an siRNA that can degrade a specific mRNA is administered to a target cell as a carrier, the mRNA can be degraded, resulting in cells with a reduced expression level of the functional protein encoded by the mRNA.

[0030] (Labeling Agent) The present disclosure also covers a labeling agent including the carrier of the present disclosure. For example, target cells can be labeled by administering to a living body a labeling substance bound to a substance that specifically recognizes target cells, etc. (a peptide, a protein, etc. that specifically binds to target cells).

[0031] (Method of Synthesizing Peptides of the Present Disclosure) The peptides of the present disclosure can be synthesized using known methods (organic synthesis, cell-based synthesis, cell-free synthesis, etc.). Considering the ease of changing the combination of each domain of the peptides of the present disclosure, it is preferable to use plasmid synthesis by an in-fusion reaction. Each domain is carried in a respective vector, and the fused plasmid is introduced into a known synthesis system (e.g., Escherichia coli system) to synthesize the desired peptide (see Figure 1).

[0032] To confirm that the carrier of the present disclosure forms a complex (particularly, a ternary complex), it is preferable to introduce a linker (e.g., a His linker) between each domain. For peptides into which a linker (e.g., a His linker) has been introduced, it can be confirmed that the carrier of the present disclosure forms a complex (particularly, a ternary complex) by a pull-down assay using a column that binds to the linker (e.g., a Ni affinity column).

[0033] (Carrier for cancer treatment of the present disclosure) The carrier for cancer treatment of the present disclosure comprises the collagen-binding membrane-permeable peptide including a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain, collagen or a collagen derivative, as described above, and a cancer therapeutic agent as the target of delivery.

[0034] (Method of Administering a Delivery Target of the Present Disclosure to a Target Cell) The method of administering a delivery target of the present disclosure to a target cell comprises the following steps: (1) administering to a mammal, including a human, a carrier comprising a collagen-binding membrane-permeable peptide comprising a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain, collagen or a collagen derivative, and the delivery target. In the method of the present disclosure, preferably, the delivery target is a cancer therapeutic agent, and the target cells / tissues are cancer cells / tissues.

[0035] (Use of the collagen-binding membrane-permeable peptide of the present disclosure for the production of a carrier) In the use of the collagen-binding membrane-permeable peptide of the present disclosure for the production of a carrier, the collagen-binding membrane-permeable peptide including the collagen-binding domain, membrane fusion domain, and membrane-permeable domain described above is used for the production of the carrier.

[0036] (Examples of peptide configurations of the present disclosure) As described above, the peptides of the present disclosure can have the following configurations, for example: CBD-S19-YGRKKRRQRRR or RKKRRQRRR or RQIKIWFQNRRMKWKK CBD-S28-YGRKKRRQRRR or RKKRRQRRR or RQIKIWFQNRRMKWKK In addition, the order of each domain (from N-terminus to C-terminus) in the above-exemplified peptides can be changed as needed. Furthermore, each domain in the above-exemplified peptides can also be replaced with each domain of another peptide to construct a peptide of the present disclosure.

[0037] The present disclosure will be described in more detail below using examples. However, the following examples should be considered as an aid to gain a concrete understanding of the present disclosure, and the scope of the present disclosure is not limited by the following examples in any way.

[0038] (Synthesis of collagen-binding membrane-permeable peptides of the present disclosure) The following collagen-binding membrane-permeable peptides of the present disclosure were synthesized using the E. coli expression system shown in Figure 1. CBD-S19-TAT: MTKKTLRT-HHHHHH-PFVIGAGVLGALGTGIGGI-HHHHHH-YGRKKRRQRRR (SEQ ID NO: 23) CBD-S19-Penetratin: MTKKTLRT-HHHHHH-PFVIGAGVLGALGTGIGGI-HHHHHH-RQIKIWFQNRRMKWKK (SEQ ID NO: 24)

[0039] Linearized pET21a(+) vector and CBD, S19, TAT, and Penetratin fragments were prepared, and the appropriate vectors and fragments were then transferred to In-Fusion. （登録商標） The plasmid was prepared by ligation using Snap Assembly Master Mix (Takara Bio). The base sequence of the prepared plasmid was confirmed by sequence analysis (contracted to AZENTA). The resulting plasmid was used to transform E. coli BL21 (DE3) strain, and the OD was reached in 1 L of LB medium containing 50 μg / ml ampicillin. 600The medium was cultured at 37°C with shaking until the pH reached approximately 0.8. After cooling on ice for 30 minutes, 0.1 mM IPTG (final concentration) was added, and then cultured at 16°C with shaking for 68–72 hours to induce protein expression. The E. coli mass was collected by centrifugation (4,700 × g, 20 minutes) and stored at -80°C. A 500 ml portion of the E. coli mass was suspended in 15 ml of lysate buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1% (v / v) Triton X-100) and sonicated. The pellet was suspended in Bind buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, 6 M guanidine hydrochloride) and shaken at 37°C for 120 minutes to denature and extract insoluble proteins. The supernatant after centrifugation was passed through a 0.45 μm filter and then applied to a HisTrap FF column. Nonspecifically bound proteins were washed away with 50 mM imidazole, and specifically bound proteins were eluted with 1 M imidazole. The eluted fraction was ultrafiltered using an Amicon filter, and the purified protein sample that passed through the filter was dialyzed and replaced with pure water. The components of each fraction were analyzed by SDS-PAGE to determine the presence and purity of the target substance. The solvent-replaced purified protein sample was frozen in liquid nitrogen and stored overnight at -80°C. The weight of the lyophilized product was measured, and the solution was adjusted to 10 mM with PBS(-).

[0040] The synthesis of the CBD-S19-TAT of the present disclosure was confirmed (Figure 2). Specifically, the successful construction of an E. coli expression system was confirmed by SDS-PAGE (Figure 2). The amino acid analysis (Figure 2) confirmed that the product purified from the E. coli expression system was CBD-S19-TAT.

[0041] The synthesis of the CBD-S19-Penetratin of the present disclosure was confirmed (Figure 3). Specifically, the successful construction of the E. coli expression system was confirmed by SDS-PAGE (Figure 3). The amino acid analysis (Figure 3) confirmed that the purified product from the E. coli expression system was CBD-S19-Penetratin.

[0042] (Complex formation of the carrier of the present disclosure) In this example, it was confirmed that the carrier of the present disclosure, "AC and collagen-binding membrane-permeable peptide" and "AC and the target of delivery (siRNA)", formed a complex. Note that, to prevent fibrillation of AC, all operations other than electrophoresis were performed in a refrigerator.

[0043] (1) Complex formation between the carrier "AC and collagen-binding membrane-permeable peptide" of the present disclosure. 250 μl of 0.1% AC (Koken Co., Ltd.) and PBS(-) were mixed and mixed by inversion for 20 minutes. CBD-S19-Penetratin was added and mixed by pipetting (final concentration: AC: 0.05%, functional peptide: 25 μM). The mixture was passed through a HisTrap FF column, washed with 5 ml of PBS(-), and then the column-bound components were eluted with 1.5 ml of Elute buffer. Proteins in each fraction were analyzed by SDS-PAGE using a 5-20% gradient gel.

[0044] (2) Complex formation between AC and delivery target (siRNA) AC and PBS(-) or siRNA prepared in PBS(-) were mixed 1:1, pipetted, and incubated for 20 minutes (final concentrations: AC: 0.05%, siRNA: 500 nM). 4 μl of 50% glycerol was added to 20 μl of each sample and mixed by pipetting. Electrophoresis was performed in the [+→-] direction on a 0.5% agarose gel at 50 V for 30 minutes. Nucleic acids were detected and observed using SYBR Gold staining, and then protein components were detected and observed using CBB staining.

[0045] Complex formation of the carrier of the present disclosure was confirmed by a pull-down assay using a Ni affinity column by agarose gel electrophoresis. The principle of the pull-down assay is shown in Figure 4. The results in Figure 5 confirmed that the AC band was shifted to the column-bound fraction by the collagen-binding membrane-permeable peptide. This confirmed that the carrier of the present disclosure formed a complex.

[0046] The results in Figure 6 confirmed that the band of the siRNA to be delivered was shifted by AC, confirming that a complex was formed between AC and the siRNA to be delivered.

[0047] The results of Figures 5 and 6 show that the collagen-binding membrane-permeable peptide, collagen or collagen derivative, and / or the target of delivery can form a complex simply by mixing them.

[0048] In this example, the concentration at which the carrier containing the target to be delivered (siRNA) does not affect cytotoxicity was confirmed.

[0049] The number of HepG2 (human hepatoma cell line)-eGFP cells was measured using a cell counter and was 1.0 × 10 5 The solution was adjusted to a concentration of 100 cells / ml, seeded in 100 μl aliquots onto a 96-well plate, and cultured overnight at 37°C and 5% CO2. AC and PBS(-) were mixed at a 1:1 ratio at 4°C and mixed by inversion for 20 minutes. siRNA (5'-CUACAACAGCCACAACGUCdTdT (SEQ ID NO: 25)-3') that suppresses eGFP expression was prepared in PBS(-) at 0.5-2 μM, and the peptide of the present disclosure was prepared at 0.02-1 mM. 5-20 μl of AC prepared in PBS(-) and 10 μl each of siRNA and the peptide of the present disclosure were added to wells containing HepG2-eGFP (final concentrations: AC: 0.005-0.02%, siRNA: 50-200 nM, functional peptide: 2-100 μM). The plate was rocked back and forth and side to side approximately 10 times to mix the medium. The cells were incubated at 37°C, 5% CO2, and observed under a microscope after 48 hours. 10 μl of CCK-8 was added to each well and the cells were cultured for 1 hour. The medium from each well reacted with CCK-8 was transferred to a new 96-well plate and then cultured using GloMax （登録商標） The absorbance was measured at 450 nm.

[0050] The cytotoxicity evaluation of the "carrier containing a target for delivery" is shown in Figure 7. The results in Figure 7 confirmed that the "carrier containing a target for delivery" of the present disclosure does not significantly affect cytotoxicity.

[0051] In this example, the efficiency of delivery of a carrier containing a delivery target (siRNA) into target cells was confirmed.

[0052] The number of HepG2-eGFP cells was measured using a cell counter and was 1.5 × 10 5 The cells / ml were adjusted, and 200 μl of each were seeded into a 48-well plate and cultured overnight at 37°C, 5% CO2. Each well was washed twice with PBS (-) and the medium was replaced with 160-200 μl. siRNA, which suppresses the expression of the target eGFP, was diluted in PBS (-). AC and various concentrations of siRNA solutions were mixed 1:1 and mixed by inversion in a refrigerator for 20 minutes. 1 μl or 2 μl of 1 mM peptide of the present disclosure was added and mixed. 40 μl of AC-siRNA-peptide of the present disclosure complex was added directly to each well after the medium was replaced, and the plate was rocked back and forth 10 times to mix (final concentrations: AC: 0.01%, siRNA: 100 or 200 nM (20 or 40 pmol), peptide of the present disclosure: 5 or 10 μM). The cells were incubated at 37°C, 5% CO2, and observed under a microscope after 48 hours. After observation, each well was washed twice with PBS (-), and the cells were detached using TrypLE Express and collected in 300 μl of PBS (-) containing 2% FBS. The cell suspension was passed through a cell strainer, and the fluorescence intensity of the cells was measured using a benchtop flow cytometer.

[0053] The results of microscopic observation are shown in Figure 8. For the "+ 10 μM CBD-S19-TAT" and "+ 10 μM CBD-S19-Penetratin" combinations, we confirmed that the fluorescence intensity decreased with increasing siRNA concentration, which suppresses the expression of the target eGFP. Figure 9 shows the results of measuring the RNAi effect of a CBD-S19-TAT-containing carrier. We confirmed that the fluorescence intensity decreased in a siRNA concentration-dependent manner. In particular, the fluorescence intensity of the "0.01% AC + 200 nM siRNA + 10 μM CBD-S19-TAT" combination was reduced to approximately one-third (18.3 / 55.1) of that of the "0.01% AC + 200 nM siRNA" combination. This indicates that CBD-S19-TAT enhanced the RNAi effect of the target, resulting from a three-fold increase in the intracellular activation efficiency of the target. Figure 10 shows the results of measuring the RNAi effect of a CBD-S19-Penetratin-containing carrier. We confirmed that the fluorescence intensity decreased in a siRNA concentration-dependent manner. In particular, the fluorescence intensity of "0.01% AC + 200 nM siRNA + 10 μM CBD-S19-Penetratin" was reduced to approximately one-sixth (9.4 / 55.1) of that of "0.01% AC + 200 nM siRNA." This indicates that CBD-S19-Penetratin enhances the RNAi effect of the target, resulting from a six-fold increase in the intracellular activation efficiency of the target.

[0054] In this example, similar to the methods described in Examples 3 and 4, H1299 cells, a type of non-small cell lung cancer cell, were used to confirm that the carrier containing the target substance (siRNA) did not affect cytotoxicity, and furthermore, the introduction of the target substance into target cells was confirmed.

[0055] The number of H1299-ZsGreen1 cells was measured using a cell counter and was 1.0 × 10 5The medium was adjusted to a concentration of 0.01% cells / ml, seeded in 100 μl aliquots into a 96-well plate, and cultured overnight at 37°C, 5% CO2. AC and PBS(-) were mixed at a 1:1 ratio at 4°C and mixed by inversion for 20 minutes. siRNA (5'-CCTCCTGCGAGAAGATCATdTdT-3' (SEQ ID NO: 35)) that suppresses ZsGreen1 expression and the peptide of the present disclosure (synthesized in Example 1) were prepared in PBS(-). AC, siRNA, and the peptide of the present disclosure prepared in PBS(-) were added to wells containing cultured H1299-ZsGreen1 cells. The final concentrations were 0.01% AC, 50-200 nM siRNA, and 2-50 μM peptide of the present disclosure. The plate was rocked back and forth approximately 10 times to mix the medium. The cells were incubated at 37°C, 5% CO2, and then observed under a microscope 48 hours later. 10 μl of CCK-8 was added to each well and incubated for 1 hour. The medium from each well reacted with CCK-8 was transferred to a new 96-well plate and then incubated with GloMax （登録商標） The absorbance at 450 nm was measured. It was confirmed that the "carrier containing a target to be delivered" of the present disclosure does not cause cytotoxicity to non-small cell lung cancer cells.

[0056] The number of H1299-ZsGreen1 cells was measured using a cell counter and was 1.5 × 10 5The cells / ml were adjusted, and 200 μl of each were seeded into a 48-well plate and cultured overnight at 37°C, 5% CO2. Each well was washed twice with PBS (-) and the medium was replaced with 160-200 μl. The siRNA (SEQ ID NO: 35) that suppresses the expression of the target protein, ZsGreen1, was diluted in PBS (-). AC and various concentrations of siRNA solutions were mixed 1:1 and mixed by inversion in a refrigerator for 20 minutes. 1 μl or 2 μl of 1 mM peptide of the present disclosure was added and mixed. 40 μl of AC-siRNA-peptide of the present disclosure complex was added directly to each well after the medium was replaced, and the plate was rocked back and forth 10 times to mix (final concentrations: AC: 0.01%, siRNA: 20, 50, 100, or 200 nM, peptide of the present disclosure: 10 μM). The cells were incubated at 37°C, 5% CO2, and observed under a microscope after 48 hours. After observation, each well was washed twice with PBS (-). Cells were then detached using TrypLE Express and collected in 300 μl of PBS (-) containing 2% FBS. The cell suspension was passed through a cell strainer, and the fluorescence intensity of the cells was measured using a benchtop flow cytometer. CBD-S19-TAT-Penetratin demonstrated the same intracellular delivery of target molecules in non-small cell lung cancer cells as in human hepatoma-derived cells.

[0057] In this example, the membrane fusion domain S28 (SEQ ID NO: 36: PFVIGAGVLGALGTGIGGITTSTQFYYK) was used to confirm the introduction of a carrier containing the target (siRNA) into target cells (H1299-ZsGreen1 cells) in the same manner as in Example 5.

[0058] According to the method of Example 1, the following collagen-binding membrane-permeable peptide of the present disclosure was synthesized and purified: CBD-S28-TAT: MTKKTLRT-HHHHHH-PFVIGAGVLGALGTGIGGITTSTQFYYK-HHHHHH-YGRKKRRQRRR (SEQ ID NO: 37)

[0059] The rate of ZsGreen1 reduction due to CBD-S28-TAT introduction was confirmed according to the method described in Example 5. CBD-S28-TAT showed the same effect as CBD-S19-TAT in introducing the target substance into target cells.

[0060] In this example, the amount of uptake of the carrier into each target cell was confirmed.

[0061] According to the method of Example 3-5, the incubation time, the presence or absence of a target (siRNA), the amount of collagen-binding membrane-permeable peptide of the present disclosure, and the amount of AC were adjusted to be as shown in Figure 11 . Figure 11 shows the percentage of cells (1 hour) into which siRNA was introduced after CBD-S28-TAT transduction in H1299 cells or HepG2 cells. Figure 11 also shows the percentage of cells (1 hour) into which siRNA was introduced after CBD-S19-TAT transduction or CBD-S19-Penetration transduction in H1299 cells or HepG2 cells. Note that 5,000 cells were counted using a flow cytometer, and the number of cells in which fluorescently labeled siRNA (target) had been translocated was extracted and calculated; therefore, the total of 5,000 cells counted was taken as 100%. As is clear from the results in Figure 11, the collagen-binding membrane-permeable peptide of the present disclosure not only rapidly delivers the target substance (siRNA) into target cells, but also delivers the target substance with high efficiency even into cells that are difficult to deliver the target substance to (e.g., HepG2 cells, with an delivery rate of 5.3% after 48 hours).

[0062] The present disclosure can provide a collagen-binding membrane-permeable peptide, and a carrier comprising the peptide and collagen or a collagen derivative.

Claims

1. A collagen-binding membrane-permeable peptide comprising a collagen-binding domain, a membrane fusion domain and a membrane-permeable domain.

2. The collagen-binding membrane-permeable peptide according to claim 1, wherein a linker is inserted between each domain.

3. The collagen binding domain is TKKTLRT (SEQ ID NO: 1), and the membrane fusion domain is X1FVIX5AX7VLX 10 ALX 13 X 14 X 15 IX 17 X 18 I (SEQ ID NO: 2) or PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36), where X1 is P or A, X5 is G or A, X7 is G or A, 10 is G or A, X 13 is G or A, X 14 is T or A, X 15 is G or A, X 17 is G or A, and X 18 is G or A, and the transmembrane domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), RQIKIWFQNRRMKWKK (SEQ ID NO: 5), GRKKRRQRRRPQ (SEQ ID NO: 6), rrrrrrrr 3. The collagen-binding membrane-permeable peptide according to claim 1, wherein the peptide is selected from the group consisting of AFLGWLGAWGTMGWSPKKKRK (SEQ ID NO: 7), AFLGWLGAWGTMGWSPKKKRK (SEQ ID NO: 8), RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9), RRLSYSRRRF (SEQ ID NO: 10), PIRRRKKLRRLK (SEQ ID NO: 11), RRQRRTSKLMKR (SEQ ID NO: 12), RRRRNRTRRNRRRVR (SEQ ID NO: 13), KMTRAQRRAAARRNRWTAR (SEQ ID NO: 14), TRRQRTRRARRNR (SEQ ID NO: 15), GRKKRRQRRRPPQ (SEQ ID NO: 16), GRRRRRRRRRPPQ (SEQ ID NO: 17), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 18), KLALKLALKLALALKLA (SEQ ID NO: 19), MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 20), or GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 21).

4. The collagen-binding membrane-permeable peptide according to claim 3, wherein the collagen-binding domain is TKKTLRT (SEQ ID NO: 1), the membrane fusion domain is PFVIGAGVLGALGTGIGGI (SEQ ID NO: 22), and the membrane-permeable domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), or RQIKIWFQNRRMKWKK (SEQ ID NO: 5).

5. The collagen-binding membrane-permeable peptide according to claim 3, wherein the collagen-binding domain is TKKTLRT (SEQ ID NO: 1), the membrane fusion domain is PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36), and the membrane-permeable domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), or RQIKIWFQNRRMKWKK (SEQ ID NO: 5).

6. A carrier comprising the collagen-binding membrane-permeable peptide according to claim 4 and collagen or a collagen derivative.

7. A carrier comprising the collagen-binding membrane-permeable peptide according to claim 5 and collagen or a collagen derivative.

8. The carrier of claim 6, further comprising an object to be carried.

9. The carrier of claim 7, further comprising an object to be carried.

10. The carrier according to claim 8, wherein the object to be transported is a protein, a peptide, a nucleic acid, and / or a low molecular weight compound.

11. The carrier according to claim 8, wherein the collagen-binding membrane-permeable peptide, the collagen or collagen derivative, and / or the target to be transported form a complex.

12. A medical device having the carrier according to claim 8 applied to its surface.

13. A cell culture device having the carrier according to claim 8 applied to a cell culture surface.

14. A drug comprising the carrier according to claim 8.

15. A carrier for cancer therapy comprising a collagen-binding membrane-permeable peptide containing a collagen-binding domain, a membrane fusion domain and a membrane-transmitting domain, collagen or a collagen derivative, and a cancer therapeutic agent as a transport target, wherein the collagen-binding domain is TKKTLRT (SEQ ID NO: 1), the membrane fusion domain is PFVIGAGVLGALGTGIGGI (SEQ ID NO: 22) or PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36), and the membrane-transmitting domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), or RQIKIWFQNRRMKWKK (SEQ ID NO: 5).

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