Collagen-binding membrane-permeable peptide and carrier containing 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

[Technical Field]

[0001] The present disclosure relates to collagen-binding membrane-permeable peptides for introducing desired proteins, peptides, nucleic acids, small molecular weight compounds, etc. into target cells, carriers containing the peptides and collagen or collagen derivatives, cell culture devices, medical devices, and drugs containing the carriers, and methods for changing the properties of target cells using the carriers. This application claims priority from Japanese Patent Application No. 2023-121675, which is incorporated herein by reference. [Background technology]

[0002] (Collagen-based carrier) Collagen is known as a sustained-release carrier for drugs (see Patent Documents 1 and 2, Non-Patent Document 1), and furthermore, kits for transferring 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 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 construction of the composite particles disclosed in that patent document differs from the construction 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 structure of the collagen to which the cell membrane-permeable peptide disclosed in the patent document is added is different from the structure of the collagen-binding membrane-permeable peptide disclosed in the present disclosure. Specifically, the cell membrane-permeable peptide disclosed in the patent document is covalently bound to a carrier. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2006-6262 [Patent Document 2] Patent 4081436 [Patent Document 3] Patent Publication No. 2014-210730 [Non-patent literature]

[0007] [Non-Patent Document 1] Cancer Res. 2004 May15;64(10):3365-70 [Non-patent document 2] http: / / www.atelocollagen.com / atelogene / japanese / index.html / Summary of the Invention [Problem to be solved by the invention]

[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. In order to address the above-mentioned problems, the present invention aims to provide a carrier that can easily introduce pharmaceuticals and the like 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 membrane-permeable peptide comprising a collagen-binding domain, a membrane fusion domain and a membrane-transducing domain. 2. The collagen-binding membrane-permeable peptide according to the preceding item 1, in which a linker is inserted between each domain. 3. The collagen binding domain is TKKTLRT (SEQ ID NO: 1); 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 18 is G or A, The transmembrane domains are 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 the preceding item 1 or 2. 4. The collagen binding domain is TKKTLRT (SEQ ID NO: 1); the fusogenic domain is PFVIGAGVLGALGTGIGGI (SEQ ID NO: 22); The transmembrane domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), or RQIKIWFQNRRMKWKK (SEQ ID NO: 5). 4. The collagen-binding membrane-permeable peptide according to the preceding item 3. 5. The collagen binding domain is TKKTLRT (SEQ ID NO: 1); the fusogenic domain is PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36); The transmembrane domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), or RQIKIWFQNRRMKWKK (SEQ ID NO: 5). 4. The collagen-binding membrane-permeable peptide according to the preceding item 3. 6. A carrier comprising the collagen-binding membrane-permeable peptide according to the preceding item 4, and collagen or a collagen derivative. 7. A carrier comprising the collagen-binding membrane-permeable peptide according to the preceding item 5, and collagen or a collagen derivative. 8. A carrier as described in the preceding paragraph 6, further including a transport object. 9. The carrier according to the preceding paragraph 7, further including a transport object. 10. The carrier according to the preceding item 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 the preceding item 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 described in the preceding paragraph 8 applied to its surface. 13. A cell culture device having the carrier described in the preceding paragraph 8 applied to the cell culture surface. 14. A drug comprising the carrier described in the preceding item 8. 15. A carrier for cancer treatment, comprising a collagen-binding membrane-permeable peptide containing a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain, collagen or a collagen derivative, and a cancer therapeutic agent as a delivery target, 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-transducing domain is YGRKKRRQRRR (SEQ ID NO: 3), RKKRRQRRR (SEQ ID NO: 4), or RQIKIWFQNRRMKWKK (SEQ ID NO: 5); Cancer therapeutic vehicles. 16. A method for administering a delivery target to a target cell, comprising the steps of: administering to a mammal, including a human, a collagen-binding membrane-permeable peptide containing a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain, collagen or a collagen derivative, and a carrier containing a target substance; wherein the collagen binding domain is TKKTLRT (SEQ ID NO: 1); 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 18 is G or A, The transmembrane domains are 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), A method for administering a delivery target to target cells. 17. The administration method 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 as a carrier, wherein the collagen binding domain is TKKTLRT (SEQ ID NO: 1); 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 18 is G or A, The transmembrane domains are 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), use. [Effects of the Invention]

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

[0012] [Figure 1] 1 shows a synthesis scheme for the collagen-binding membrane-permeable peptide of the present disclosure. [Figure 2] Synthesis confirmation of CBD-S19-TAT of the present disclosure. [Figure 3] Confirmation of the synthesis of CBD-S19-Penetratin of the present disclosure. [Figure 4] Schematic diagram of pull-down assay using Ni affinity column. [Figure 5] Confirmation of complex formation between the carrier of the present disclosure (atelocollagen (AC) and collagen-binding membrane-permeable peptide) [Figure 6] Confirmation of complex formation between AC and the target (siRNA). [Figure 7] Cytotoxicity evaluation of the "carrier containing a delivery target" of the present disclosure. [Figure 8] Evaluation of RNA interference (RNAi) by vehicles of the present disclosure. [Figure 9] Confirmation of the RNAi effect of a carrier containing the CBD-S19-TAT of the present disclosure. [Figure 10] Confirmation of the RNAi effect of a carrier containing CBD-S19-Penetratin of the present disclosure. [Figure 11]Confirmation of the amount of uptake in each cell of the delivery target contained in the delivery vehicle of the present disclosure.

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

[0014] The "carrier" of the present disclosure has the function of transporting 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 simply transports a target substance to a cell, but also has one or more of the following functions: sustained release, long-term retention in target cells, specific localization around target cells, and protection of the target substance from in vivo degradation.

[0015] Peptides of the present disclosure The peptide of the present disclosure includes at least a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain. If necessary, a known linker (particularly a His linker (consisting of 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive His)) may be introduced between each domain. In addition, the order of each domain in the peptide (from the N-terminus to the C-terminus) is not particularly limited, but examples include the following: Collagen-binding domain - membrane fusion domain - membrane translocation domain Collagen-binding domain - membrane-transducing domain - membrane-fusion domain Membrane fusion domain - collagen binding domain - membrane translocation domain Membrane fusion domain - membrane transduction domain - collagen binding domain Membrane transduction domain - collagen binding domain - membrane fusion domain Membrane transduction 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 have 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 containing each of the domains shown below in which 1 to 5 (1, 2, 3, or 4) amino acids have been substituted, deleted, inserted, and / or added, and which have substantially the same activity as the peptides. In addition, from the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide (amino acid) when introducing mutations or substitutions into the peptide (amino acid), 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.) can be easily envisioned.

[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. An example is TKKTLRT (SEQ ID NO: 1).

[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 sequences 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 13is 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 details are as follows: 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 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 a 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-transducing domain) The transmembrane 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" that is generally used in the medical, cosmetic, industrial, and food fields. It is preferable to use soluble or solubilized collagen as collagen. Soluble collagen is soluble in acidic or neutral water or salt solution. Solubilized collagen includes enzyme-solubilized collagen solubilized by enzymes and alkali-solubilized collagen solubilized by 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 collagen extracted from vertebrates is preferred, more preferably from mammals, birds, or fish, and even more preferably from mammals or birds with a high denaturation temperature. Any type of collagen may be used, but types I to V are preferred in terms of the amount present in animal bodies. Specific examples include type I collagen extracted with an acid from mammalian dermis, and more preferred examples include type I collagen extracted with an 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 less tyrosine residues per 1000 residues is more preferred. The preferred collagen or collagen derivative of the present disclosure is atelocollagen.

[0020] (Method for producing a carrier according to the present disclosure) The delivery body of the present disclosure can be produced by a method known per se, by mixing the peptide of the present disclosure, the collagen or collagen derivative of the present disclosure, and the object to be delivered. This provides the following effects by forming a complex (particularly a ternary complex). (1) It plays a role in protecting the nucleic acid carrier from nucleases in the body. (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 peptides of the present disclosure are 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, a biocompatible material, an additive, and the like. Examples of biocompatible materials include gelatin, fibrin, albumin, hyaluronic acid, heparin, chondroitin sulfate, chitin, chitosan, alginic acid, pectin, agarose, hydroxyapatite, polypropylene, polyethylene, polydimethylsiloxane, or polymers of glycolic acid, lactic acid, or amino acids, 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 the pH at 6 to 8 or to maintain isotonicity with cells. Furthermore, the carrier of the present disclosure may be in a solid form or a solution form. When the carrier of the present disclosure is in a solid form, it is loaded onto the desired cells as it 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 it can be used for drugs (particularly cancer treatment drugs), medical devices, cell culture devices, cosmetic raw materials, fertilizers, food additives, labeling agents, etc.

[0023] (Method of administering the carrier of the present disclosure) The method of administering the carrier of the present disclosure to a living body (an animal including a human, particularly a mammal including a human) may be oral administration, injection, eye drop, nasal drop, pulmonary administration, or absorption through the skin, with injection being preferred. 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. Examples include systemic administration of the carrier of the present disclosure by intravenous injection (infusion, etc.), or local administration by injection into the affected area (cancer cells, cancer tissue, etc.).

[0024] (Transportation target) The "subject to be delivered" in the present disclosure is not particularly limited, but examples thereof include proteins, peptides, nucleic acids, low molecular weight compounds, and the like.

[0025] (protein) Examples include, but are not limited to, enzymes, agonists / antagonists of target receptors, receptors themselves, antibodies, etc. (peptide) These include low molecular weight proteins with enzymatic activity, partial synthesis of functional proteins, and agonists and antagonists for target receptors, but are not limited to these. It is not something that can be done. (Low molecular compound) Examples of such drugs include, but are not limited to, low molecular weight drugs such as anticancer drugs that are specifically effective in killing tumor cells, and drugs that enhance or suppress the physiological activity of cells. (nucleic acid) In the present disclosure, the "nucleic acid to be delivered" may be a polynucleotide or an oligonucleotide, and 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 strand, it may be the coding strand or the non-coding strand. "Nucleic acid" includes DNA derivatives or RNA derivatives, and these derivatives refer to nucleic acids having phosphorothioate bonds or nucleic acids in which the phosphate moiety, sugar moiety, or base moiety of internucleotides has 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 a plasmid DNA or a virus, it is preferably in a form 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 containing elements that enable integration into a chromosome.

[0026] (Medical devices or cell culture equipment) The present disclosure also covers a medical device or cell culture device having a carrier of the present disclosure applied to its surface. When the carrier of the present disclosure is applied to a solid surface and then contacted with target cells, the efficiency of delivery of the target substance is improved compared to when the carrier of the present disclosure is added dropwise from above the target cells. The medical devices of the present disclosure include artificial organs, more specifically, artificial blood vessels, medical device stents for reinforcing blood vessels, adhesive sheets, and artificial hearts. Examples of the cell culture device of the present disclosure include a petri dish, a flask, a 96-well microplate, a three-dimensional culture carrier, and the like that are commonly used in cell culture experiments.

[0027] (Method for investigating the function of a gene or protein in a target cell) The use of the delivery vehicle of the present disclosure makes it easy to examine the function of a gene or protein in a target cell. For example, a useful method is to examine the function of a gene by introducing a plasmid DNA incorporating the gene whose function is to be examined into the cell and expressing the gene, or to examine the function of the gene by introducing an siRNA nucleic acid that suppresses the expression of the gene whose function is to be examined into the cell and suppressing the expression of the gene. In a specific measurement method, a carrier of the present disclosure is mixed with a plasmid DNA expressing a gene of interest, an adenovirus vector, or an siRNA nucleic acid suppressing the expression of the gene of interest, and then coated and aligned on a solid phase of a culture plate. After drying and immobilizing the coated carrier on the solid phase, cells are seeded and cultured on the plate for several days. The coated carrier is efficiently introduced into the cells that adhere to the coated area, 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 cells (gene expression level), or the type and amount of protein produced by the cells.

[0028] (Method for screening carriers capable of treating diseases) The carrier of the present disclosure can be used 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 carrier 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 carrier is dried and fixed on the solid phase, cells are seeded and cultured on the plate for several days. The effect of nucleic acids can be analyzed by changes in cell phenotype, cell death, cell proliferation, intracellular gene expression patterns, and the types and amounts of proteins produced.

[0029] (Method for changing the properties of target cells using a carrier) The carrier of the present disclosure can be used to change the properties of target cells. For example, by administering an antagonist that suppresses the activity of a specific intracellular receptor to a target cell as the delivery target, the activity of the receptor in the target cell can be reduced (the properties of the target cell can be changed) compared to target cells that have not been administered with the antagonist. In addition, by administering siRNA capable of degrading specific mRNA to target cells, the mRNA can be degraded, resulting in cells with reduced expression of the functional protein encoded by that 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 (e.g., a peptide or protein 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 methods known per se (organic synthesis, cell-based synthesis, cell-free synthesis, etc.). Considering the ease of changing the combination of each domain of the peptide of the present disclosure, it is preferable to use plasmid synthesis by in-fusion reaction. Each domain is carried in a vector, and the fused plasmid can be introduced into a known synthesis system (e.g., E. 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 by a pull-down assay using a column that binds to the linker (e.g., a Ni affinity column) that the carrier of the present disclosure forms a complex (particularly a ternary complex).

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

[0034] (Method of administering the delivery target of the present disclosure to target cells) The method of administering the delivery target of the present disclosure to target cells comprises the following steps. (1) A step of administering to a mammal, including a human, a collagen-binding membrane-permeable peptide containing a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain, collagen or a collagen derivative, and a carrier containing a target to be transported. In the method of the present disclosure, the object to be delivered is preferably a cancer therapeutic agent, and the target cells / tissues are preferably 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 as a carrier, the collagen-binding membrane-permeable peptide including the collagen-binding domain, membrane fusion domain, and membrane-permeable domain described above is used to produce the carrier.

[0036] (Examples of peptide configurations of the present disclosure) As described above, the peptide 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 the N-terminus to the C-terminus) in the above-exemplified peptides can be changed as necessary. Furthermore, each domain in the above-exemplified peptides can be replaced with each domain of other peptides to construct the peptides 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. Example 1

[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 FIG. 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 Escherichia coli BL21 (DE3) strain, and the OD was reached in 1 L of LB medium containing 50 μg / ml ampicillin. 600 The 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 CBD-S19-TAT of the present disclosure was confirmed to have been synthesized (Figure 2). In detail, the successful construction of the E. coli expression system was confirmed by SDS-PAGE in Figure 2. The amino acid analysis in Figure 2 confirmed that the purified product from the E. coli expression system was CBD-S19-TAT.

[0041] It was confirmed that the CBD-S19-Penetratin of the present disclosure has been synthesized (Figure 3). In detail, the successful construction of the E. coli expression system was confirmed by SDS-PAGE in Figure 3. The amino acid analysis in Figure 3 confirmed that the purified product from the E. coli expression system was CBD-S19-Penetratin. Example 2

[0042] (Complex formation of the carrier of the present disclosure) In this example, it was confirmed that the carriers of the present disclosure, "AC and collagen-binding membrane-permeable peptide" and "AC and the target of delivery (siRNA)" formed complexes. Note that, to prevent AC from becoming fibrous, all operations other than electrophoresis were carried out 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 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 the target (siRNA) AC and PBS(-) or siRNA prepared in PBS(-) were mixed at a 1:1 ratio, 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 on a 0.5% agarose gel at 50 V for 30 minutes in the [+→-] direction. Nucleic acids were detected and observed using SYBR Gold staining, followed by protein detection and observation using CBB staining.

[0045] The complex formation of the carrier of the present disclosure was confirmed by a pull-down assay using a Ni affinity column and 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. This confirmed that the AC and the target (siRNA) formed a complex.

[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. Example 3

[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 5The cells / ml were adjusted, and 100 μl of each was seeded into 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 the target to be delivered" is shown in Figure 7. The results in FIG. 7 confirmed that the "carrier containing a target for delivery" of the present disclosure does not significantly affect cytotoxicity. Example 4

[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 5The cells / ml were adjusted, and 200 μl of each were seeded onto 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 directly added 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 "+ 10 μM CBD-S19-TAT" and "+ 10 μM CBD-S19-Penetratin," it was confirmed that the fluorescence intensity decreased when the concentration of siRNA that suppresses the expression of eGFP, the target of delivery, was increased. 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 "0.01% AC + 200 nM siRNA + 10 μM CBD-S19-TAT" was reduced to approximately one-third (18.3 / 55.1) of that of "0.01% AC + 200 nM siRNA." This indicates that CBD-S19-TAT enhanced the RNAi effect of the carrier, resulting from a three-fold increase in the intracellular activation efficiency of the carrier. Figure 10 shows the results of measuring the RNAi effect of a carrier containing CBD-S19-Penetratin. 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 1 / 6 (9.4 / 55.1) compared to "0.01% AC + 200 nM siRNA." This indicates that CBD-S19-Penetratin improved the RNAi effect of the carrier, resulting from a six-fold increase in the intracellular activation efficiency of the carrier. Example 5

[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 5 The culture medium was adjusted to a concentration of 100 cells / ml, and 100 μl of the medium was seeded onto a 96-well plate and cultured overnight at 37°C in 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 of each were 0.01% for AC, 50-200 nM for siRNA, and 2-50 μM for the peptide of the present disclosure. The plate was shaken back and forth about 10 times to mix the medium. The plate was 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 plate was 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. It was confirmed that the "carrier containing a target for delivery" of the present disclosure does not exert cytotoxicity on non-small cell lung cancer cells.

[0056] The number of H1299-ZsGreen1 cells was measured using a cell counter and was 1.5 × 10 5 The culture medium was adjusted to a concentration of 100 cells / ml, and 200 μl of the medium was seeded onto a 48-well plate and cultured overnight at 37°C in 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 mixed by rocking back and forth 10 times (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 (-), 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. CBD-S19-TAT·Penetratin showed the same intracellular delivery effect of the target substance in non-small cell lung cancer cells as in human hepatoma-derived cells. Example 6

[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 peptides of the present disclosure were synthesized and purified. CBD-S28-TAT:MTKKTLRT-HHHHHH-PFVIGAGVLGALGTGIGGITTSTQFYYK-HHHHHH-YGRKKRRQRRR (SEQ ID NO: 37)

[0059] According to the method described in Example 5, the rate of reduction of ZsGreen1 due to introduction of CBD-S28-TAT was confirmed. CBD-S28-TAT showed the same effect of delivering the target substance into the target cells as CBD-S19-TAT. Example 7

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

[0061] According to the method of this Example 3-5, the culture time, the presence or absence of the target to be delivered (siRNA), the amount of the collagen-binding membrane-permeable peptide of the present disclosure, and the amount of AC were adjusted to be as shown in FIG. The percentage of cells (1 hour) into which siRNA was introduced into H1299 cells or HepG2 cells after CBD-S28-TAT transfection was confirmed. The results are shown in FIG. Furthermore, the percentage of cells (1 hour) into which siRNA was introduced into H1299 cells or HepG2 cells by CBD-S19-TAT transfection or CBD-S19-Penetration transfection was confirmed. The results are shown in Figure 11. Note that 5,000 cells were counted using a flow cytometer, and the number of cells in which fluorescently labeled siRNA (the target of delivery) had been transferred into the cells was extracted and calculated, so 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). [Industrial Applicability]

[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-transducing domain, the collagen binding domain is TKKTLRT (SEQ ID NO: 1); the membrane fusion domain is PFVIGAGVLGALGTGIGGI (SEQ ID NO: 22); the transmembrane domain is YGRKKRRQRRR (SEQ ID NO: 3) or RQIKIWFQNRRMKWKK (SEQ ID NO: 5), In addition, a His linker is inserted between each domain. Collagen-binding membrane-permeable peptide.

2. 2. The collagen-binding membrane-permeable peptide according to claim 1, wherein the His linker is composed of 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive His residues.

3. A carrier comprising the collagen-binding membrane-permeable peptide according to claim 1 or 2 and collagen or a collagen derivative. Here, the carrier is any one of the following (1) to (7): (1) Carrier for transporting screening candidate substances (2) A carrier that protects the target from biodegradation and releases the target into target cells. (3) Drug delivery vehicle (4) A carrier having a sustained release effect, a retention effect in target cells, a specific localization effect around target cells, or an effect of protecting the delivered substance from in vivo degradation. (5) Carrier for cancer treatment (6) Transporter for proteins, peptides, nucleic acids or low molecular weight compounds (7) Carriers of substances that induce enhancement or suppression of physiological activity

4. The carrier according to claim 3 , further comprising a target to be carried.

5. The carrier according to claim 4, wherein the object to be transported is a protein, a peptide, a nucleic acid, and / or a low molecular weight compound.

6. The carrier according to claim 4 , wherein the collagen-binding membrane-permeable peptide, the collagen or collagen derivative, and / or the target to be transported form a complex.

7. A medical device having the carrier according to claim 4 applied to its surface.

8. A cell culture device having the carrier according to claim 4 applied to a cell culture surface.

9. A drug comprising the carrier according to claim 4.

10. A carrier for cancer treatment, comprising a collagen-binding membrane-permeable peptide including a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain, collagen or a collagen derivative, and a cancer therapeutic agent as a delivery target, 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-transducing domain is YGRKKRRQRRR (SEQ ID NO: 3) or RQIKIWFQNRRMKWKK (SEQ ID NO: 5); Cancer therapeutic vehicles.

11. A medical device comprising a collagen-binding membrane-permeable peptide containing a collagen-binding domain, a membrane fusion domain, and a membrane-transducing domain, collagen or a collagen derivative, and a drug; 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), the transmembrane domain is YGRKKRRQRRR (SEQ ID NO: 3) or RQIKIWFQNRRMKWKK (SEQ ID NO: 5), In addition, a His linker is inserted between each domain. Medical equipment.

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