Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/08—Materials for coatings
  • A61L31/10—Macromolecular materials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

• 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 a target cell, a carrier comprising said peptide and collagen or a collagen derivative, a cell culture device, a medical device, or a drug comprising said carrier, and a method for changing the properties of a target cell using said carrier.
• 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, a kit for introducing nucleic acid into target cells is commercially available (see Non-Patent Document 2).
• Patent Document 1 discloses a "cell transfection array for nucleic acid introduction comprising atelocollagen, a nucleic acid introduction agent and a nucleic acid.”
• Patent Document 2 discloses "complex particles containing atelocollagen and a desired nucleic acid, the nucleic acid being single-stranded or double-stranded and the major axis of the particle being 100 ⁇ m or less.”
• the composition of the composite particles disclosed in that patent differs from the composition of the carriers of the present disclosure.
• Patent Document 3 discloses a "carrier comprising collagen or a collagen derivative having a cell membrane-permeable peptide added thereto.”
• the structure of collagen to which a cell membrane-permeable peptide is added disclosed in the patent document is different from the structure of the collagen-binding membrane-permeable peptide disclosed in the present disclosure. More specifically, the cell membrane-permeable peptide disclosed in the patent document is covalently bound to a carrier.
• an object of the present invention is to provide a carrier that can easily introduce a pharmaceutical agent or the like into a cell.
• a collagen-binding membrane-permeable peptide containing a collagen-binding domain, a membrane fusion domain, and a membrane-permeable domain, as well as a carrier containing said peptide and collagen or a collagen derivative, can solve the above problems, and thus completed the present disclosure.
• a collagen-binding membrane-permeable peptide comprising a collagen-binding domain, a membrane fusion domain and a membrane-permeable domain.
• the collagen binding domain is TKKTLRT (SEQ ID NO: 1); the membrane fusogenic domain is X1FVIX5AX7VLX10ALX13X14X15IX17X18I (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, X10 is G or A , X13 is G or A, X14 is T or A, X15 is G or A, X17 is G or A, and X18 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), rrrrrrrrr (SEQ ID NO: 7
• 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), RKKRRQRRR (SEQ ID NO: 4), or RQIKIWFQNRRMKWKK (SEQ ID NO: 5).
• the collagen binding domain is TKKTLRT (SEQ ID NO:1);
• the membrane fusion 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).
• a carrier comprising the collagen-binding membrane-permeable peptide according to the preceding item 4, and collagen or a collagen derivative.
• a carrier comprising the collagen-binding membrane-permeable peptide according to the above item 5, and collagen or a collagen derivative.
• 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.
• a cell culture instrument having a cell culture surface coated with the carrier according to the preceding item 8.
• a carrier for cancer treatment comprising a collagen-binding membrane-permeable peptide including a collagen-binding domain, a membrane fusion domain, and a membrane-permeable 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)
• the membrane transduction 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 to which a substance to be delivered is administered comprising the steps of: Administering a collagen-binding membrane-permeable peptide including a collagen-binding domain, a membrane fusion domain, and a membrane-permeable domain, collagen or a collagen derivative, and a carrier including a target to a mammal, including a human; wherein the collagen binding domain is TKKTLRT (SEQ ID NO:1), the membrane fusion domain is X1FVIX5AX7VLX10ALX13X14X15IX17X18I ( 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, X10 is G or A , X13 is G or A, X14 is T or A, X15 is G or A, X17 is G
• a method for administering a delivery target to a target cell 17.
• the object to be delivered is a cancer therapeutic agent and the target cells are cancer cells.
• a collagen-binding membrane-permeable peptide comprising a collagen-binding domain, a membrane fusion domain and a membrane-permeable domain as a carrier, comprising: wherein the collagen binding domain is TKKTLRT (SEQ ID NO:1), the membrane fusion domain is X1FVIX5AX7VLX10ALX13X14X15IX17X18I ( 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, X10 is G or A , X13 is G or A, X14 is T or A, X15 is G or A, X17 is G or A, and
• the collagen-binding membrane-permeable peptide of the present disclosure and a carrier comprising said peptide and collagen or a collagen derivative have one or more of the following effects.
• (2) A complex (particularly a ternary complex) is formed simply by mixing a collagen-binding membrane-permeable peptide, collagen or a collagen derivative, and an object to be transported.
• (4) The target substance can be delivered with high efficiency even into target cells into which the target substance is difficult to deliver.
• RNAi RNA interference
• 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 comprising the peptide and collagen or a collagen derivative (hereinafter, may be referred to as the “carrier of the present disclosure”)," a “cell culture instrument having the carrier applied to a cell culture surface (hereinafter, may be referred to as the “cell culture instrument of the present disclosure”),” and a “pharmaceutical agent comprising the carrier (hereinafter, may be referred to as the "pharmaceutical agent of the present disclosure”).
• the “carrier” of the present disclosure has the function of transporting the target to a cell ⁇ a target cell (a cell into which the target is introduced) ⁇ . Furthermore, the carrier of the present disclosure not only transports the target to a cell, but also has one or more of the following functions: sustained release, long-term retention in the target cell, specific localization around the target cell, and protection of the target from degradation in the body.
• 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 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.
• each domain in the peptide is not particularly limited, but examples include the following: Collagen-binding domain-membrane fusion domain-transmembrane domain Collagen-binding domain-membrane transmembrane domain-membrane fusion domain Membrane fusion domain-collagen-binding domain-transmembrane domain Membrane fusion domain-membrane transmembrane domain-collagen-binding domain Me ...
• the peptides of the present disclosure also include protected derivatives, glycosylated derivatives, acylated derivatives, or acetylated derivatives of peptides comprising each of the domains shown below.
• the peptides of the present disclosure also include peptides that have 90-99% (95-99%, 97-99%, 98-99%) or more identity to peptides containing each of the domains shown below and have substantially the same activity as said peptides.
• 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.
• amino acid amino acid
• 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.
• 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.
• TKKTLRT SEQ ID NO: 1
• TKKTLRT SEQ ID NO: 1
• the membrane fusion domain is not particularly limited as long as it is a sequence that allows the peptide of the present disclosure to fuse to a cell membrane.
• the following sequences can be exemplified: X 1 FVIX 5 AX 7 VLX 10 ALX 13 X 14 X 15 IX 17 X 18 I (Arrangement number 2) PFVIGAGVLGALGTGIGGITTSTQFYYK (SEQ ID NO: 36)
• X1 is P or A
• X5 is G or A
• X7 is G or A
• X10 is G or A
• X13 is G or A
• X14 is T or A
• X15 is G or A
• X17 is G or A
• X18 is G or A.
• 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 domain which is a peptide described in SEQ ID NO: 26 to SEQ ID NO: 34 and
• the membrane-transducing domain is not particularly limited as long as it is a sequence that allows the peptide of the present disclosure to penetrate a cell membrane.
• 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) GWTLNSAGYLLGKINLKALAALA
• collagen or collagen derivative refers to any "collagen or collagen derivative” that is generally used in the medical, cosmetic, industrial, and food fields.
• collagen it is preferable to use soluble or solubilized 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 collagen extracted from mammals, birds, and fish, and more preferably collagen extracted from mammals and birds with high denaturation temperatures. Any type of collagen may be used, but types I to V are preferred in terms of the amount present in the animal body.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.
• telocollagen from which highly antigenic telopeptides have been enzymatically removed or atelocollagen produced by genetic engineering is preferred, 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.
• the carrier of the present disclosure can be produced by a method known per se, by mixing the peptide of the present disclosure, collagen or collagen derivative of the present disclosure, and the object to be transported.
• the formation of a complex has the following effects. (1) It plays a role in protecting the nucleic acid carrier from nucleases in the body.
• the carrier 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 the cell (cytoplasm).
• the collagen-binding membrane-permeable peptides of the present disclosure are preferably not covalently bound in the carrier.
• 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.
• 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.
• additives include isotonicity agents, pH regulators, and pain-relieving 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.
• the carrier of the present disclosure may be in a solid or 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.
• the uses of the carrier of the present disclosure are not particularly limited, but it can be used for drugs (particularly cancer treatments), medical instruments, cell culture instruments, cosmetic raw materials, fertilizers, food additives, labeling agents, etc.
• the method of administering the carrier of the present disclosure to a living body may be any of oral administration, injection, eye drop, nasal drop, pulmonary administration, and absorption through the skin, preferably injection.
• the administration site can be selected according to the disease, but it can also be directly placed at a necessary site during surgery (e.g., cancer cells, cancer tissue).
• 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.).
• 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.
• Examples of the target receptor include, but are not limited to, agonists and antagonists of enzymes and target receptors, the receptors themselves, and antibodies.
• (peptide) Examples of such substances include, but are not limited to, low molecular weight proteins having enzyme activity, synthesis of portions of functional proteins, and agonists and antagonists for target receptors.
• Low molecular weight compounds 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.
• the "nucleic acid to be delivered” may be a polynucleotide or an oligonucleotide, and may be a DNA or an RNA molecule.
• a DNA molecule it may be a plasmid DNA, a cDNA, a genomic DNA, or a synthetic DNA.
• both DNA and RNA may be double-stranded or single-stranded.
• a single strand it may be a coding strand or a non-coding strand.
• Nucleic acid includes DNA derivatives or RNA derivatives, and the derivatives refer to nucleic acids having phosphorothioate bonds, or nucleic acids in which the phosphate moiety, sugar moiety, or base moiety of the internucleotide has been chemically modified to avoid enzymatic degradation.
• Nucleic acid also includes viruses such as adenoviruses and retroviruses.
• the nucleic acid 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 a gene of interest, such as a promoter, or containing elements that enable integration into a chromosome.
• a vector containing elements necessary for expression of a gene of interest such as a promoter, or containing elements that enable integration into a chromosome.
• the present disclosure is also directed to a medical device or cell culture device having a carrier of the present disclosure applied to its surface.
• a medical device or cell culture device having a carrier of the present disclosure applied to its surface.
• the medical device of the present disclosure includes an artificial organ, more specifically, an artificial blood vessel, a medical device for reinforcing a blood vessel, a stent, an adhesive sheet, or an artificial heart.
• the cell culture apparatus of the present disclosure includes 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.
• Method for investigating the function of a gene or protein in a target cell By using the carrier of the present disclosure, the function of a gene or protein in a target cell can be easily examined. For example, a method of examining the function of a gene by introducing a plasmid DNA incorporating the gene whose function is to be examined into a cell and expressing the gene, or a method of examining the function of a gene by introducing an siRNA nucleic acid that suppresses the expression of the gene whose function is to be examined into a cell and suppressing the expression of the gene, is useful.
• a plasmid DNA expressing a gene whose function is to be elucidated, an adenovirus vector, or an siRNA nucleic acid suppressing the expression of a gene whose function is to be elucidated is mixed with the carrier of the present disclosure, and the carrier is applied to a solid phase of a culture plate and aligned. After the applied carrier is dried and fixed on the solid phase, cells are seeded and cultured on the plate for several days. The applied carrier is efficiently introduced into the cells attached to the applied part, and expresses the gene whose function is to be examined or suppresses its expression for a long period of time. After several days, the function of the targeted gene can be clarified by examining the cell proliferation rate, morphology (phenotype), state of gene expression in the cell (gene expression level), or the type and amount of protein produced by the cell.
• a candidate substance e.g., nucleic acid
• a candidate substance whose therapeutic effect on a disease is to be examined 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 the nucleic acid can be analyzed by changes in cell phenotype, cell death, cell proliferation, patterns of gene expression within cells, and the types and amounts of proteins produced.
• 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, the target cell can have lower receptor activity (the properties of the target cell can be changed) compared to a target cell to which the antagonist has not been administered.
• siRNA capable of degrading a specific mRNA to a target cell it is possible to obtain cells in which the mRNA is degraded and the expression level of the functional protein encoded by that mRNA is reduced.
• the present disclosure also covers a labeling agent including the carrier of the present disclosure.
• a labeling agent including the carrier of the present disclosure.
• a carrier in which a labeling substance is bound to a substance that specifically recognizes target cells or the like a peptide, a protein, or the like that specifically binds to target cells
• a carrier in which a labeling substance is bound to a substance that specifically recognizes target cells or the like can be administered to a living body to label the target cells.
• the peptide 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 is introduced into a known synthesis system (e.g., E. coli system) to synthesize the desired peptide (see FIG. 1).
• a known synthesis system e.g., E. coli system
• a linker e.g., a His linker
• a linker e.g., a His linker
• the cancer treatment vehicle of the present disclosure comprises the above-described collagen-binding membrane-permeable peptide including a collagen-binding domain, a membrane fusion domain and a membrane-permeable domain, collagen or a collagen derivative, and a cancer therapeutic agent as the target to be delivered.
• a method for administering a delivery target of the present disclosure to a target cell comprises the following steps. (1) A step of administering to a mammal, including a human, a collagen-binding membrane-permeable peptide including a collagen-binding domain, a membrane fusion domain, and a membrane-permeable domain, collagen or a collagen derivative, and a carrier including a target to be transported.
• the object to be delivered is preferably a cancer therapeutic agent
• the target cells/tissues are preferably cancer cells/cancer tissues.
• the collagen-binding membrane-permeable peptide of the present disclosure for the production of a carrier
• the collagen-binding membrane-permeable peptide including the above-described collagen-binding domain, membrane fusion domain, and membrane-permeable domain is used for the production of the carrier.
• the peptide of the present disclosure can have the following configurations. CBD-S19-YGRKKRRQRRR or RKKRRQRRR or RQIKIWFQNRRMKWKK CBD-S28-YGRKKRRQRRR or RKKRRQRRR or RQIKIWFQNRRMKWKK
• the order of each domain (from the N-terminus to the C-terminus) in the above-exemplified peptides can be changed as necessary.
• each domain in the above-exemplified peptides can be changed to each domain of other peptides to construct the peptides of the present disclosure.
• a linearized pET21a(+) vector and each fragment of CBD, S19, TAT and Penetratin were prepared, and the appropriate vector and fragment were connected with In-Fusion (registered trademark) Snap Assembly Master Mix (Takara Bio) to prepare a plasmid.
• the base sequence of the prepared plasmid was confirmed by sequence analysis (contracted to AZENTA).
• the obtained plasmid was used to transform E. coli BL21 (DE3) strain, and it was cultured with shaking at 37 ° C in 1 L of LB medium containing 50 ⁇ g / ml ampicillin until OD 600 reached about 0.8.
• the pellets obtained after centrifugation were 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 ⁇ m filter and then through a HisTrap FF.
• Nonspecifically bound proteins were washed with 50 mM imidazole, and specifically bound proteins were eluted with 1 M imidazole.
• the eluted fraction was ultrafiltered with Amicon, 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 purified protein samples after solvent exchange were frozen in liquid nitrogen and stored at -80°C overnight.
• the weight of the lyophilized product was measured and adjusted to 10 mM with PBS(-).
• CBD-S19-TAT of the present disclosure was synthesized ( Figure 2).
• the successful construction of the E. coli expression system was confirmed by SDS-PAGE in Figure 2.
• the purified product from the E. coli expression system was confirmed to be CBD-S19-TAT by amino acid analysis in Figure 2.
• the 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 FIG.
• the results in FIG. 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.
• HepG2 human hepatoma cell line
• eGFP cells The number of HepG2 (human hepatoma cell line)-eGFP cells was measured with a cell counter and adjusted to 1.0 ⁇ 10 5 cells/ml, and 100 ⁇ l of each solution was seeded on a 96-well plate and cultured overnight at 37° C., 5% CO 2.
• AC and PBS(-) were mixed at a ratio of 1:1 at 4° C. and mixed by inversion for 20 minutes.
• siRNA (5'-CUACAACAGCCACAACGUCdTdT (SEQ ID NO: 25)-3') that suppresses the expression of eGFP was prepared in PBS(-) at 0.5-2 ⁇ M, and the peptide of the present disclosure was prepared at 0.02-1 mM.
• the cytotoxicity evaluation of the “carrier containing the target to be delivered” is shown in FIG. From the results in FIG. 7, it was confirmed that the "carrier containing a transport target" of the present disclosure does not significantly affect cytotoxicity.
• the efficiency of delivery of the carrier containing the target substance (siRNA) into the target cells was confirmed.
• the number of HepG2-eGFP cells was measured using a cell counter, adjusted to 1.5 ⁇ 10 5 cells/ml, and 200 ⁇ l of each solution was seeded on a 48-well plate and cultured overnight at 37°C, 5% CO 2. Each well was washed twice with PBS (-) and the medium was replaced with 160-200 ⁇ l. siRNA that suppresses the expression of eGFP, the target of delivery, was diluted with PBS (-). AC and siRNA solutions of each concentration were mixed 1:1 and mixed by inversion in a refrigerator for 20 minutes. 1 ⁇ l or 2 ⁇ l of 1 mM peptide of the present disclosure was added and mixed.
• AC-siRNA-peptide of the present disclosure complex 40 ⁇ l of AC-siRNA-peptide of the present disclosure complex was directly added to each well after replacing the medium, and the plate was shaken back and forth and left and right 10 times to mix (final concentration; AC: 0.01%, siRNA: 100 or 200 nM (20 or 40 pmol), peptide of the present disclosure: 5 or 10 ⁇ M).
• 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 ⁇ l 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 improved the RNAi effect of the carrier, and is due to a three-fold increase in the intracellular activation efficiency of the carrier by CBD-S19-TAT.
• the results of measuring the RNAi effect of a carrier containing CBD-S19-Penetratin are shown in Figure 10. It was confirmed that the fluorescence intensity decreased depending on the siRNA concentration. In particular, it was confirmed that the fluorescence intensity of "0.01% AC + 200 nM siRNA + 10 ⁇ M CBD-S19-Penetratin" was reduced to about 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, and is due to a six-fold increase in the intracellular activation efficiency of the carrier by CBD-S19-Penetratin.
• H1299 cells which are non-small cell lung cancer cells, were used to confirm that the carrier containing the target (siRNA) did not affect cytotoxicity, and furthermore, the introduction of the target into the target cells was confirmed.
• the number of H1299-ZsGreen1 cells was measured using a cell counter, and the cells were adjusted to 1.0 ⁇ 10 5 cells/ml. 100 ⁇ l of the cells were seeded onto a 96-well plate and cultured overnight at 37° C., 5% CO 2 . 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 in which H1299-ZsGreen1 cells were cultured.
• the final concentrations of each were 0.01% for AC, 50-200 nM for siRNA, and 2-50 ⁇ M for the peptide of the present disclosure.
• the plate was rocked back and forth about 10 times to mix the medium.
• the plates were incubated at 37°C, 5% CO2 , and observed under a microscope after 48 hours.
• 10 ⁇ l of CCK-8 was added to each well and cultured for 1 hour.
• the medium from each well reacted with CCK-8 was transferred to a new 96-well plate, and the absorbance at 450 nm was measured using GloMax (registered trademark) . It was confirmed that the "carrier containing a delivery target" of the present disclosure does not cause cytotoxicity to non-small cell lung cancer cells.
• the number of H1299-ZsGreen1 cells was measured using a cell counter, and the cells were adjusted to 1.5 ⁇ 10 5 cells/ml. 200 ⁇ l of the cells were seeded onto a 48-well plate and cultured overnight at 37° C., 5% CO 2 . Each well was washed twice with PBS (-) and the medium was replaced with 160-200 ⁇ l. siRNA (SEQ ID NO: 35) that suppresses the expression of ZsGreen1, the target of delivery, was diluted with PBS (-). AC and siRNA solutions of each concentration were mixed 1:1 and mixed by inversion in a refrigerator for 20 minutes. 1 ⁇ l or 2 ⁇ l of 1 mM peptide of the present disclosure were added and mixed.
• AC-siRNA-peptide of the present disclosure complex 40 ⁇ l of AC-siRNA-peptide of the present disclosure complex was directly added to each well after replacing the medium, and the plate was shaken back and forth and left and right 10 times to mix (final concentration; AC: 0.01%, siRNA: 20, 50, 100 or 200 nM, peptide of the present disclosure: 10 ⁇ M).
• the plates 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 with TrypLE Express and collected in 300 ⁇ l 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.
• 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.
• 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)
• CBD-S28-TAT showed the same effect of delivering the target molecule into the target cells as CBD-S19-TAT.
• FIG. 11 shows the percentage of cells (1 hour) into which siRNA was introduced into H1299 cells or HepG2 cells by CBD-S28-TAT transfection. Furthermore, the percentage of cells into which siRNA was introduced (1 hour) in H1299 cells or HepG2 cells by CBD-S19-TAT transfection or CBD-S19-Penetration transfection was confirmed. The results are shown 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 into cells into which it is difficult to deliver the target substance (e.g., HepG2 cells, with an introduction rate of 5.3% after 48 hours).
• the present disclosure can provide a collagen-binding membrane-permeable peptide, as well as a carrier that contains the peptide and collagen or a collagen derivative.

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