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WO2021040022A1 - Nouveau peptide pénétrant les cellules et utilisation associée - Google Patents

Nouveau peptide pénétrant les cellules et utilisation associée Download PDF

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Publication number
WO2021040022A1
WO2021040022A1 PCT/JP2020/032760 JP2020032760W WO2021040022A1 WO 2021040022 A1 WO2021040022 A1 WO 2021040022A1 JP 2020032760 W JP2020032760 W JP 2020032760W WO 2021040022 A1 WO2021040022 A1 WO 2021040022A1
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WIPO (PCT)
Prior art keywords
membrane
amino acid
residue
peptide
permeable peptide
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Ceased
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PCT/JP2020/032760
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English (en)
Japanese (ja)
Inventor
生彦 中瀬
桃子 小吹
未来 片山
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University Public Corporation Osaka
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University Public Corporation Osaka
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Priority to JP2021543082A priority Critical patent/JPWO2021040022A1/ja
Publication of WO2021040022A1 publication Critical patent/WO2021040022A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to a membrane-permeable peptide found from saporin toxin.
  • the present invention also relates to a complex containing the membrane-permeable peptide and a target molecule, and a method for producing the same.
  • the present invention comprises a membrane-permeable peptide or a reagent containing the complex, a pharmaceutical preparation containing the complex, a method for improving the membrane permeability of a target molecule, a method for transfecting a nucleic acid molecule, and a cell for target molecule. Regarding the method of introducing into.
  • the lipid bilayer which is the basic structure of the cell membrane, is composed of amphipathic phospholipid molecules, and has a structure in which the hydrophilic part is exposed and the hydrophobic part is put inside. Since the cell membrane is hydrophobic as a whole and hardly allows ions and hydrophilic substances to pass through, extremely small substances such as water molecules and carbon dioxide molecules or non-polar substances can penetrate the cell membrane, but amino acids, nucleic acids, Sugars and proteins cannot permeate. Therefore, some ingenuity is required to send high molecular weight compounds and nanoparticles from the outside of the cell to the inside of the cell. Due to recent advances in medicine, many protein preparations and nucleic acid medicines have been developed. A drug delivery system (DDS) for efficiently and directly delivering these to the affected area has been attracting attention and research is underway.
  • DDS drug delivery system
  • a method of introducing a physiologically active substance into a cell using a polypeptide called a membrane-permeable peptide is often used.
  • the membrane-permeable peptide is a peptide having the property of penetrating the cell membrane and translocating into the cell. This property can be used to deliver substances that normally cannot penetrate cell membranes into cells.
  • a complex in which a membrane-permeable peptide and a substance to be introduced into a cell are bound (covalently and non-covalently bound) is prepared and added to a cell culture solution, whereby the substance is introduced into the cell.
  • Various studies have been conducted on the application of this membrane-permeable peptide as a new delivery carrier for pharmaceuticals into cells.
  • Typical membrane-permeable peptides include TAT peptide, which is a basic peptide derived from TAT protein derived from human immunodeficiency virus type 1 (HIV-1), and oligoarginine (most of the sequences with about 1-20 bases).
  • membrane-permeable peptides such as TAT peptides and oligoarginines are non-permeable due to their positive charge, such as being trapped by interacting with serum components and being adsorbed on the surface of glass such as test tubes. It is known that specific adsorption occurs.
  • the present inventors focused on saporin toxin in order to solve the above problems. Then, the present inventors highly efficiently transfer the peptide (RFR peptide: RFRYIQNLVTKNFPNKF) having the amino acid sequence represented by SEQ ID NO: 1 possessed by the saporin toxin into the cell, and the non-specific adsorption is reduced.
  • the present invention has been completed by finding that it is a product.
  • the present invention provides a membrane-permeable peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which 1 or more and 10 or less amino acid residues are deleted, substituted or added in the amino acid sequence.
  • the present invention also provides a complex containing the above-mentioned membrane-permeable peptide and a target molecule.
  • the present invention provides a method for producing the above-mentioned complex, which comprises binding the above-mentioned membrane-permeable peptide to a target molecule.
  • the present invention provides a reagent containing the above-mentioned membrane-permeable peptide or the above-mentioned complex.
  • the present invention provides a pharmaceutical preparation containing the above complex. Furthermore, the present invention provides a method for improving the membrane permeability of a target molecule containing the above-mentioned membrane-permeable peptide and the target molecule. Furthermore, the present invention provides a method for transfecting a nucleic acid molecule, which comprises mixing the above membrane-permeable peptide with a nucleic acid molecule. Further, in the present invention, the amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 7 to SEQ ID NO: 16, or an amino acid residue of 1 to 10 is deleted, substituted or added in the amino acid sequence. Provided is a method for introducing a target molecule into a cell using a membrane-permeable peptide consisting of an amino acid sequence.
  • a membrane-permeable peptide with reduced non-specific adsorption is provided. It can be used for DDS of drugs and research reagents.
  • 6 is an image showing a cell fluorescence signal of HeLa cells exposed to fluorescently labeled peptide-containing media 2, 5 and 6.
  • 6 is a graph showing the cell viability of HeLa cells exposed to fluorescently labeled peptide-containing media 1 and 3. It is a graph which shows the cell fluorescence amount of CHO-K1 cell and CHO-A745 cell exposed to the fluorescently labeled peptide containing medium 3. It is a graph which shows the cell fluorescence amount of CHO-K1 cell and CHO-A745 cell exposed to the fluorescently labeled peptide containing medium 1. It is an observation image by a confocal laser scanning microscope of HeLa cells to which QD-SA-B-RFR was added.
  • amino acid residues are described in one-letter notation according to a conventional method. Further, in the present specification, the amino acid sequence of the peptide is described from left to right from the N-terminal to the C-terminal according to a conventional method. Further, in the present specification, “N-terminal” and “N-terminal side” mean the amino acid residue shown on the leftmost side of the amino acid sequence represented by each SEQ ID NO: and the amino acid sequence shown on the left side, respectively, for convenience. .. Similarly, “C-terminal” and “C-terminal side” mean the amino acid residue shown on the rightmost side of the amino acid sequence represented by each SEQ ID NO: and the amino acid sequence shown on the right side, respectively.
  • the amino acid residue in the present invention is not limited to the L form, but may be the D form.
  • the membrane-permeable peptide of the present invention is a polypeptide having an amino acid sequence (RFR sequence) represented by SEQ ID NO: 1 (RFRYIQNLVTKNFPNKF) possessed by a saporin toxin (molecular weight of about 30,000: SEQ ID NO: 2).
  • the membrane-permeable peptide of the present invention is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted or added in the amino acid sequence. You may.
  • the number of amino acid residues to be deleted, substituted or added is 1 or more and 10 or less, preferably 1 or more and 5 or less.
  • the number of amino acid residues to be deleted, substituted or added may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the substitution is preferably a conservative substitution.
  • Conservative substitution refers to a substitution in which properties such as acidity and basicity do not substantially change between the original amino acid residue and the amino acid residue after the substitution. Specifically, substitution between F, W, Y, substitution between L, I, V, substitution between K, R, H, substitution between D, E, substitution between S, T. Point to.
  • the amino acid residue to be deleted or substituted may be any amino acid residue, but amino acid residues other than lysine residue, arginine residue and phenylalanine residue. Is preferable.
  • the amino acid sequence represented by SEQ ID NO: 1 is the 1st arginine residue, the 2nd phenylalanine residue, the 3rd arginine residue, the 11th lysine residue, and the 13th phenylalanine residue. , 16th lysine residue and 17th phenylalanine residue are preferably not deleted.
  • the amino acid sequence represented by SEQ ID NO: 1 is the 1st arginine residue, the 2nd phenylalanine residue, the 3rd arginine residue, the 11th lysine residue, the 13th phenylalanine residue, and the 16th. It is preferable that the lysine residue and the 17th phenylalanine residue are not substituted. Examples of substitutions for amino acid residues other than lysine residue, arginine residue and phenylalanine residue include, for example, substitution of amino acid residues 4 to 10, 12, 14 and 15 as alanine residues, 6th.
  • substitutions that make glutamine residues alanine residues or substitutions that make the 7, 12 and 15th asparagine residues alanine residues may be selected from substitutions that make glutamine residues alanine residues or substitutions that make the 7, 12 and 15th asparagine residues alanine residues.
  • substitution with an alanine residue has been exemplified, but the present invention is not limited to this, and an amino acid residue other than alanine may be substituted (for example, substitution with a valine residue, a leucine residue, an isoleucine residue, etc.).
  • the amino acid residues substituted in the amino acid sequence represented by SEQ ID NO: 1 may be lysine residues, arginine residues and phenylalanine residues in the amino acid sequence represented by SEQ ID NO: 1.
  • the substitution of the amino acid residue is, for example, the substitution of the first arginine residue of the amino acid sequence represented by SEQ ID NO: 1 as an alanine residue, the substitution of the third arginine residue as an alanine residue, and the eleventh substitution.
  • substitution of lysine residue to alanine residue substitution of 16th lysine residue to alanine residue, substitution of 2nd phenylalanine residue to alanine residue, 13th phenylalanine residue to alanine residue It may be selected from the substitution to make the 17th phenylalanine residue an alanine residue.
  • substitution with an alanine residue has been exemplified, but the present invention is not limited to this, and an amino acid residue other than alanine may be substituted (for example, substitution with a valine residue, a leucine residue, an isoleucine residue, etc.). ).
  • the number of amino acid residue substitutions may be one or plural. Even if these sequences are replaced, a membrane-permeable peptide with reduced non-specific adsorption to a test tube or the like can be obtained.
  • the membrane-permeable peptide of the present invention includes SEQ ID NO: 7 (AFRYIQNLVTKNFPNKF), SEQ ID NO: 8 (RFAYIQNLVTKNFPNKF), SEQ ID NO: 9 (RFRYIQNLVTANFPNKF), SEQ ID NO: 10 (RFRYIQNLVTKNFPNAF), SEQ ID NO: 11 (RARYIQNLVTKNFPNAF), SEQ ID NO: 12 (RARYIQNLVTKNFPNKF), SEQ ID NO: 12 , SEQ ID NO: 13 (RFRYIQNLVTKNFPNKA), SEQ ID NO: 14 (RFRAAAAAAAKAFAAKF), SEQ ID NO: 15 (RFRYIANLVTKNFPNKF) or SEQ ID NO: 16 (RFRYIQALVTKAFPAKF).
  • SEQ ID NO: 7 AFRYIQNLVTKNFPNKF
  • SEQ ID NO: 8 RAYIQNLVTKNFPNKF
  • SEQ ID NO: 9 RFRYIQNLVTANFPNKF
  • the membrane-permeable peptide of the present invention is derived from an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted or added in the amino acid sequence represented by any one of SEQ ID NO: 7 to SEQ ID NO: 16. It may be a membrane-permeable peptide.
  • the number of amino acid residues to be deleted, substituted or added is 1 or more and 10 or less, preferably 1 or more and 5 or less.
  • the number of amino acid residues to be deleted, substituted or added may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the substitution is preferably a conservative substitution. The conservative replacement is as described above.
  • the membrane-permeable peptide of the present invention contains amino acid residues of 1 to 35 at at least one of the N-terminal and C-terminal of the amino acid sequence represented by any one of SEQ ID NO: 1 and SEQ ID NO: 7 to SEQ ID NO: 16. It may be a polypeptide to which an additional sequence is added.
  • the additional sequence may contain any amino acid residue, but preferably contains a residue selected from a lysine residue, an arginine residue and a phenylalanine residue.
  • the number of amino acid residues in the additional sequence may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35.
  • the amino acid sequence can be analyzed by using an analysis program generally used by those skilled in the art. For example, GENETYX-WIN Ver. 7 can be used for amino acid sequence analysis. Similar to the amino acid sequence, the base sequence can be analyzed and searched by an analysis program generally used by those skilled in the art.
  • Membrane permeability in the present invention refers to the ability of a substance to permeate a cell membrane.
  • Membrane permeability can be rephrased as the ability of a substance to move from extracellular to intracellular.
  • the means of penetrating the cell membrane is not particularly limited, and the substance is cell-permeated by, for example, endocytosis, macropinocytosis, via a transmembrane protein (eg, a receptor), direct membrane permeation by physical properties, or a combination thereof. You may move to the inside.
  • Membrane permeability in the present invention may include endosome prolapse. Endosome prolapse means that a substance that is taken up by endocytosis and exists in the endosome escapes from the endosome to the cytosol.
  • Membrane permeation also includes transfection of nucleic acid molecules.
  • the membrane-permeable peptide of the present invention By adding the membrane-permeable peptide of the present invention to cells, the membrane-permeable peptide can permeate the cell membrane and migrate into the cell. Further, by binding or associating the membrane-permeable peptide of the present invention with the target molecule described later, the target molecule can be incorporated into the cell together with the membrane-permeable peptide. Therefore, the membrane-permeable peptide of the present invention can be used to deliver the target molecule into the cell.
  • the membrane-permeable peptide of the present invention can be added to cells at any concentration.
  • the membrane-permeable peptide of the present invention has the ability to efficiently permeate the cell membrane even at a final concentration of 10 ⁇ M or less in the solution or medium after addition, and has the ability to permeate the cell membrane with high efficiency even at a concentration of 2 ⁇ M or less.
  • the membrane-permeable peptide of the present invention has the ability to efficiently permeate the cell membrane even at a low concentration of 500 nM or less in a solution or medium.
  • the final concentration of the membrane-permeable peptide in the solution or medium is 1M, 500 ⁇ M, 100 ⁇ M, 50 ⁇ M, 20 ⁇ M, 10 ⁇ M, 9 ⁇ M, 8 ⁇ M, 7 ⁇ M, 6 ⁇ M, 5 ⁇ M, 4 ⁇ M, 3 ⁇ M, 2 ⁇ M, 1 ⁇ M, 500nM, 400. It can be added to cells so as to be 300nM, 200nM, 100nM, 50nM, 10nM, 5nM, 1nM.
  • the membrane-permeable peptide of the present invention can be obtained by a method generally used by those skilled in the art to obtain a polypeptide.
  • a peptide solid phase synthesis method such as Fmoc synthesis method or Boc synthesis method can be used.
  • a vector having a polynucleotide encoding the membrane-permeable peptide sequence of the present invention may be prepared and introduced into a host to express the membrane-permeable peptide to obtain a membrane-permeable peptide.
  • the host is not particularly limited as long as it can be cultured and can express a polypeptide, and for example, cultured cells, non-pathogenic Escherichia coli, yeast, actinomycetes, algae, lactic acid bacteria, Bacillus subtilis and the like can be used.
  • the culture conditions can be appropriately adjusted according to the host and expression system.
  • the medium is not particularly limited as long as the culture target can grow.
  • the medium may be solid or liquid, but is preferably liquid.
  • the culturing method is not particularly limited as long as the culturing target can grow. For example, static culture, shaking culture, anaerobic culture and the like can be used.
  • the culture conditions can be appropriately adjusted according to the host and expression system.
  • the membrane-permeable peptide-containing synthetic solution or culture obtained by the above method can be purified by a method generally used by those skilled in the art to obtain a purified membrane-permeable peptide.
  • the purification method is not particularly limited as long as a membrane-permeable peptide can be obtained.
  • the culture supernatant containing the membrane-permeable peptide is subjected to centrifugation or filter filtration to remove cells and solids, and then the cells and solids are removed. It can be purified by combining filtration or concentration with an ultrafiltration filter, salting out, chromatography using a column, adsorption with activated charcoal, and the like.
  • the column to be used is not particularly limited as long as it can be separated from other proteins and impurities without inactivating the enzyme.
  • Examples of the column include a column for anion exchange chromatography, a column for cation exchange chromatography, a column for hydrophobic interaction chromatography, a column for gel filtration chromatography and the like.
  • the crudely purified or purified membrane-permeable peptide can be detected by a method without particular limitation as long as the membrane-permeable peptide contained in the sample can be separated and detected. For example, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry or a method using electrophoresis such as SDS-PAGE is used.
  • the membrane-permeable peptide may be in a solution state or in a dry state.
  • the solvent is not particularly limited, and examples thereof include water, a sodium phosphate solution, and a sodium acetate solution.
  • Examples of the method for drying the membrane-permeable peptide include a method of drying it by subjecting it to a freeze-dryer.
  • the present invention also provides a complex containing the membrane-permeable peptide and a target molecule.
  • the target molecule is a substance that exerts some action or effect on the cell by being transported into the cell by the membrane-permeable peptide of the present invention.
  • the target molecule include pharmaceutical compounds.
  • the pharmaceutical compound is not particularly limited as long as it has some medicinal activity on living organisms and cells. For example, low molecular weight compounds having a molecular weight of 10,000 or less, antibodies, nucleic acids, vectors, oligosaccharides, lipopolysaccharides, and sugar chains. , Peptides, cyclic peptides, glycopeptides, proteins, glycoproteins, fluorescent substances and the like.
  • Proteins include not only naturally occurring proteins but also intrinsically disordered proteins such as recombinant proteins.
  • Nucleic acids include not only naturally occurring nucleic acids but also artificial nucleic acids such as siRNA, morpholino oligos, and phosphorothioates.
  • Fluorescent materials include fluorescent dyes such as fluorescein isothianate (FITC), rhodamine, Alexa Fluor®, fluorescent proteins such as Green Fluorescent Protein (GFP) or quantum dots. Quantum dots may have other molecules added, such as Qdot® 525-Streptavidin Conjugate and Qdot® 605 Biotin Conjugate. These target molecules may be labeled with a radioisotope. Examples of the radioactive isotope include 125 I, 14 C, 32 P and the like.
  • the membrane-permeable peptide and the target molecule may be in a state where they are associated in a solution or in a form in which they are bound.
  • the binding mode is not particularly limited, and examples thereof include covalent bonds (peptide bonds, disulfide bonds, etc.) and non-covalent bonds (ionic bonds, hydrogen bonds, etc.).
  • the complex can cause the target molecule to act on the cell, for example, by penetrating the cell membrane and translocating to the cytosol.
  • the membrane-permeable peptide and the target molecule may be directly bound or indirectly bound to the target molecule. Examples of direct bonds include covalent bonds.
  • Specific examples thereof include linking the peptide and the target molecule with a cross-linking agent such as maleimide, and obtaining a fusion protein of the peptide and the target molecule by gene recombination.
  • Indirect binding includes, for example, binding via a combination of biotins and avidins.
  • Biotins include biotin and biotin analogs such as desthiobiotin.
  • Avidins include avidins and analogs of avidins such as streptavidin and tamavidin®.
  • An example of indirect binding between a membrane-permeable peptide and a target molecule is the indirect binding of a membrane-permeable peptide and a quantum dot.
  • biotin to the membrane-permeable peptide and streptavidin to the quantum dots
  • the membrane-permeable peptide and quantum dots can be indirectly bound via the binding of biotin and streptavidin.
  • the present invention also provides a method for producing the complex described above, which comprises binding the membrane-permeable peptide to a target molecule.
  • a complex can be obtained by binding the target molecule to the membrane-permeable peptide obtained by the above method.
  • the obtained complex can be suitably used for reagents and pharmaceutical formulations.
  • the target molecule is in the form of a peptide or protein, it may be produced by a vector designed so that the membrane-permeable peptide and the target molecule are bound from the beginning.
  • a histidine tag for purification may be added to the membrane-permeable peptide or the target molecule.
  • a method for binding the membrane-permeable peptide to the target molecule a method generally used by those skilled in the art can be used.
  • a binding method for example, an organic synthesis method such as an addition reaction or a coupling reaction, or an enzymatic reaction can be used for binding.
  • the target molecule is a peptide, it may be synthesized in a state of being bound to the membrane-permeable peptide from the beginning by using a peptide solid phase synthesis method such as Fmoc synthesis method or Boc synthesis method.
  • the present invention also provides a reagent containing the above-mentioned membrane-permeable peptide or the above-mentioned complex.
  • Reagents may include uses such as research reagents and reagents for analysis / analysis.
  • Examples of the research reagent and the reagent for analysis / analysis include a cell fluorescence reagent and a protein-inducing reagent.
  • the cell fluorescence reagent the state of the target cultured cells can be analyzed by adding a membrane-permeable peptide to which a fluorescent dye has been added to the cultured cells and measuring the fluorescence.
  • the protein-inducing reagent can express a specific protein in the cell by adding a membrane-permeable peptide to which a specific sequence is added to the cultured cells.
  • the reagent may contain additives as long as its properties are not significantly impaired.
  • Additives include surfactants (eg sodium citrate, sodium dodecyl sulfate, etc.), preservatives (methyl or propyl p-hydroxybenzoate, sorbic acid, tocopherol, etc.), pH regulators (sodium hydrogen carbonate, potassium carbonate, etc.) Examples include citrates, acetates, etc.), antioxidants (vitamin C, vitamin E, etc.), chelating agents (disodium edetate, sodium citrate, sodium metaphosphate, etc.).
  • the reagent of the present invention may contain other measurement reagents and the like together with the above-mentioned membrane-permeable peptide or the above-mentioned complex.
  • the present invention also provides a pharmaceutical preparation containing the above complex.
  • the pharmaceutical preparation may further contain a drug having other pharmacological actions in addition to the above-mentioned membrane-permeable peptide or the above-mentioned complex.
  • the pharmaceutical product may be a liquid or a solid. Examples of the form of the pharmaceutical preparation include tablets, pills, powders, granules, capsules, and liquids. Additives may be appropriately added to the pharmaceutical preparation as long as the pharmacological activity is not impaired.
  • an additive it can be appropriately selected from pharmaceutically acceptable additives.
  • excipients Arabic rubber, gelatin, sorbitol, tragant, hydroxypropyl cellulose, ethyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc.
  • fillers lactose, sugar, corn starch, calcium phosphate, sorbitol, mannitol, glycine, etc.
  • disintegrants for example, excipients (Arabic rubber, gelatin, sorbitol, tragant, hydroxypropyl cellulose, ethyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc.
  • fillers lactose, sugar, corn starch, calcium phosphate, sorbitol, mannitol, glycine, etc.
  • Non-aqueous excipients includemond oil, fractionated coconut oil or glycerin, propylene glycol, polyethylene glycol, oily esters such as ethyl alcohol, etc.), storage Agents (methyl or propyl p-hydroxybenzoate, sorbitol, etc.), chelating agents (disodium edetate, sodium citrate, sodium metaphosphate, etc.), pH adjusters (sodium hydrogencarbonate, potassium carbonate, etc.), thickeners Examples include (Arabic rubber, methylcellulose, etc.), antioxidants (vitamin C, vitamin E, etc.), coating agents (titanium oxide, iron sesquioxide, etc.).
  • the present invention is the use of a membrane-permeable peptide for producing the above-mentioned reagent or pharmaceutical preparation, wherein the membrane-permeable peptide is represented by any one of SEQ ID NO: 1, SEQ ID NO: 7 to SEQ ID NO: 16. Also provided is the use of a membrane-permeable peptide consisting of an amino acid sequence, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted or added in the amino acid sequence.
  • the membrane-permeable peptide used may be a simple substance or may be in the form of the above-mentioned complex.
  • the membrane-permeable peptide used may be one synthesized by a peptide solid phase synthesis method or the like, or may be one produced by the host described above.
  • the membrane-permeable peptide can be mixed with water and / or the above-mentioned additive to produce the above-mentioned reagent or pharmaceutical preparation.
  • the amount of the membrane-permeable peptide used in the reagent or the pharmaceutical preparation is not particularly limited. For example, if the reagent and the pharmaceutical preparation are in the form of a solution, the peptide can be added so that the final concentration of the membrane-permeable peptide in the solution is in the range of 1M to 1nM.
  • the final concentration of the membrane-permeable peptide in the solution is 1M, 500 ⁇ M, 100 ⁇ M, 50 ⁇ M, 20 ⁇ M, 10 ⁇ M, 9 ⁇ M, 8 ⁇ M, 7 ⁇ M, 6 ⁇ M, 5 ⁇ M, 4 ⁇ M, 3 ⁇ M, 2 ⁇ M, 1 ⁇ M, 500nM, 400. , 300nM, 200nM, 100nM, 50nM, 10nM, 5nM, 1nM can be added.
  • 100 to 0.1 parts by weight of the membrane-permeable peptide can be added to 100 parts by weight of the substance excluding the membrane-permeable peptide in the reagent or the pharmaceutical preparation.
  • 100 parts by weight, 90 parts by weight, 80 parts by weight, 70 parts by weight, and 60 parts by weight of the membrane-permeable peptide are added to 100 parts by weight of the substance excluding the membrane-permeable peptide in the reagent or pharmaceutical preparation.
  • the present invention also provides a method for improving the membrane permeability of a target molecule, which comprises binding the membrane-permeable peptide to a target molecule.
  • the binding step is not particularly limited as long as the membrane-permeable peptide and the target molecule can coexist and can bind to each other, but a binding mode that does not inhibit the activity of the target molecule is preferable.
  • the present invention is an amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 7 to SEQ ID NO: 16, or an amino acid in which 1 to 10 amino acid residues are deleted, substituted or added in the amino acid sequence. Also provided is a method of introducing a target molecule into a cell using a membrane-permeable peptide consisting of a sequence.
  • the membrane-permeable peptide of the present invention can be used for any cell. For example, it can be appropriately selected from cells derived from human or non-human mammals. It is considered that the membrane-permeable peptide of the present invention is not affected by the sugar chain of the cell when it permeates the cell membrane.
  • the membrane-permeable peptide can permeate into the cell even if the cell does not have a sugar chain on the cell surface, and the target molecule can be introduced into the cell.
  • This is a property different from the TAT peptide, which reduces the efficiency of introduction into cells for cells having no sugar chain on the cell membrane surface. Due to this property, the membrane-permeable peptide of the present invention can select cells to be introduced without being affected by the presence or absence of sugar chains on the cell membrane surface.
  • the membrane-permeable peptide of the present invention is unlikely to cause non-specific adsorption to the surface of a solid phase such as glass, polystyrene, polypropylene, polyimide or silicone resin. Therefore, the loss of the membrane-permeable peptide due to non-specific adsorption can be suppressed, and the amount of the membrane-permeable peptide used can be suppressed. In addition, the membrane-permeable peptide of the present invention can be easily dissolved in water. Furthermore, since the membrane-permeable peptide of the present invention is less adsorbed on the cover glass or the like, non-specific fluorescence when observing cells using the membrane-permeable peptide combined with a fluorescent substance can be reduced. .. Therefore, the background is reduced when observing with a microscope or the like, and the cells of interest can be easily seen.
  • the present invention also provides a method for transfecting a nucleic acid molecule, which comprises mixing the membrane-permeable peptide with a nucleic acid molecule.
  • the membrane-permeable peptide of the present invention can translocate even a small amount of nucleic acid into the cell. This makes it possible to transfect cells even with a small amount of nucleic acid, which was difficult to introduce with conventional transfection methods.
  • the nucleic acid molecule is a protein expression vector
  • the present invention is obtained by transfecting the vector containing a nucleic acid having a base sequence encoding GFP with a membrane-permeable peptide and measuring the fluorescence derived from GFP. The effect of transfection can be confirmed.
  • the nucleic acid to be introduced is not particularly limited, but a nucleic acid as the above-mentioned pharmaceutical compound or a plasmid vector can be used.
  • the cell-penetrating peptide of the present invention may be used alone or in combination with a commercially available transfection reagent when mixed with a nucleic acid molecule.
  • the transfection reagent to be combined is not particularly limited, but a reagent based on the principle of forming a complex with a nucleic acid molecule and being taken up by cells by endocytosis is preferable.
  • the transfection method comprises mixing a membrane-permeable peptide, a nucleic acid molecule, and a transfection reagent to form a complex.
  • the complex is taken up by cells by endocytosis, it is considered that the complex or nucleic acid molecule escapes from the endosome by the action of the membrane-permeable peptide of the present invention.
  • the fluorescently labeled peptide is prepared by mixing the cysteine side chain in the peptide sequence with Alexa Fluor 488 C5 maleimide sodium salt (Invitrogen, Eugene: Alexa488) with the peptide synthesized and purified by the above Fmoc solid phase method, and at room temperature. It was prepared by reacting for 3 hours. The prepared fluorescently labeled peptide was purified by reverse phase high performance liquid chromatography in the same manner as described above, and the molecular weight was confirmed by a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer.
  • Example 1 Exposure of fluorescently labeled peptides to cells (cultured cells) Human cervical cancer-derived HeLa cells (Riken BRC Cell Bank: HeLa cells) were used as exposure targets for the fluorescently labeled peptide. HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) in a 100 mm cell culture dish under 37 ° C. and 5% CO 2 conditions, 2- It was subcultured and maintained every 3 days.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • HeLa cells (2 ⁇ 10 5 cells, 2 mL) 24 hours in DMEM medium 35 mm glass based dishes (37 °C, 5% CO 2 ) after and the cells washed with DMEM medium (500 [mu] L, 3 times).
  • DMEM medium containing 500nM RFR-Alexa488 Fluorescently labeled peptide-containing medium 1
  • FBS Fluorescently labeled peptide-containing medium 2
  • DMEM medium containing 500nM TAT-Alexa488 Fluorescently labeled peptide-containing medium 3
  • DMEM medium containing 500nM TAT-Alexa488, 10% FBS: Fluorescently labeled peptide-containing medium 4 was prepared, added to each of the washed cells, and cultured for 30 minutes (medium volume 200 ⁇ L, 37 ° C., 5% CO 2 ).
  • HeLa cells cultured in each fluorescently labeled peptide-containing medium are added to nuclear staining reagent (Hoechst 33342, 5 ⁇ g / mL, 200 ⁇ L) , incorporated into cells for 20 minutes (37 ° C, 5% CO 2 ), and then in DMEM medium. The cells were washed (500 ⁇ L, 3 times). The cell fluorescence signal of these HeLa cells was observed using a confocal laser scanning microscope (FV1200, Olympus).
  • nuclear staining reagent Hoechst 33342, 5 ⁇ g / mL, 200 ⁇ L
  • HeLa cells (7.0 ⁇ 10 4 cells, 1 mL) were cultured in 24-well microplates for 24 hours (37 ° C, 5% CO 2 ), and then the cells were washed with DMEM medium (200 ⁇ L, 3 times). Fluorescently labeled peptide-containing media 1 to 4 were added to the washed cells and cultured for 30 minutes (medium volume 300 ⁇ L, 37 ° C., 5% CO 2 ).
  • HeLa cells cultured in each fluorescently labeled peptide-containing medium were washed with heparin (0.5 mg / mL) -containing phosphate buffered saline (PBS) (200 ⁇ L, 3 times). After washing, trypsin (0.1 g / L) treatment (200 ⁇ L, 37 ° C., 10 minutes, 5% CO 2 ) was performed to peel off the cells. After collecting the peeled cells, they were centrifuged at 4 ° C. and 200 ⁇ g. After centrifugation, the supernatant was removed and PBS (400 ⁇ L) was added to disperse the cells. Centrifugation was performed again at 4 ° C.
  • heparin 0.5 mg / mL
  • PBS phosphate buffered saline
  • FIGS. 1 and 2 The results of measuring the cell fluorescence signal and the amount of cell fluorescence of the fluorescently labeled peptide-containing media 1 and 3 are shown in FIGS. 1 (confocal laser scanning microscope observation) and FIG. 2 (flow cytometer measurement).
  • the left panel of FIG. 1 shows the green fluorescence of Alexa 488
  • the center panel shows the blue fluorescence of Hoechst
  • the right panel is a merged image of these.
  • the lower right bar of each image represents the length of 20 ⁇ m in the image. From FIG. 1, it was clearly recognized that RFR-Alexa488 was taken up into HeLa cells in the image when RFR-Alexa488 was used.
  • Example 2 Cytotoxicity evaluation of fluorescently labeled peptide Since the RFR peptide is derived from saporin toxin, it was verified whether the RFR peptide itself is not toxic. Toxicity evaluation of fluorescently labeled peptides on cells was performed by measuring the cell viability of fluorescently labeled peptide-exposed cells. Cell viability was evaluated by WST-1 (4- [3- (4-iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio] -1,3-benzenedisulfonate) assay. It was.
  • HeLa cells (1.2 x 10 4 cells, 100 ⁇ L) were cultured in 96 well microplates (IWAKI) for 24 hours (37 ° C, 5% CO 2 ), then cell-washed in DMEM medium (50 ⁇ L, 3 times) and fluorescently labeled.
  • Peptide-containing medium 1 or 3 was added and cultured for 30 minutes (medium volume 50 ⁇ L, 37 ° C., 5% CO 2 ).
  • WST-1 reagent (10 ⁇ L) was added and cultured for 30 minutes (37 ° C., 5% CO 2 ), and the absorbance (450 nm and 620 nm (background)) was measured.
  • a microplate reader (Thermo Scientific Multiskan) was used to measure the absorbance.
  • DMEM containing no fluorescently labeled peptide was used instead of the medium containing the fluorescently labeled peptide, and the cell viability of the control was set to 100%.
  • the results of the cytotoxicity evaluation test of the fluorescently labeled peptide are shown in FIG. Compared with the control, RFR-Alexa488 maintained cell viability and RFR-Alexa488 was found to be non-toxic to cells. From this, it was found that the RFR peptide is a saporin-derived peptide that is toxic to cells, but is a non-toxic and safe peptide.
  • the RFR peptide has less non-specific adsorption and is highly efficiently taken up by cells.
  • the RFR peptide is highly efficiently taken up into cells even under low concentration conditions such as 500 nM in solution. This indicates that RFR peptides are excellent carriers for drug delivery systems that transport target molecules into cells.
  • the low non-specific adsorption of RFR peptides is also excellent in terms of improving the visibility of fluorescence observation of cells.
  • Example 3 Glycan Dependence of Membrane Permeable Peptides
  • Basic peptides such as TAT peptides are known to be highly dependent on sugar chains on the surface of cell membranes during intracellular translocation.
  • sugar chain dependence of the RFR peptide of the present invention was examined.
  • the above RFR-Alexa488 and TAT-Alexa488 were used.
  • a fluorescently labeled peptide-containing medium 8 in which TAT-Alexa488 was suspended was prepared so as to be.
  • Commercially available Chinese hamster ovary-derived CHO-K1 cells (American Type Culture Collection (ATCC)) and CHO-A745 cells (ATCC) lacking all surface sugar chains of CHO-K1 cells were used as exposure targets. ..
  • CHO-K1 cells and CHO-A745 cells (140,000 cells each) were added to 1 ml of 10% FBS-containing F-12 medium in a 24-well microplate and cultured for 24 hours.
  • Fluorescent-labeled peptide-containing medium 7 and fluorescent-labeled peptide-containing medium 8 were added to the cultured CHO-K1 cells and CHO-A745 cells, respectively, and cultured for 30 minutes (medium volume 600 ⁇ L, 37 ° C.). Then, the same operation as in Example 1 was performed on the cultured CHO-K1 cells and CHO-A745 cells, and the amount of cell fluorescence was measured using a flow cytometer (10,000 living cells (3 times)). , Excitation 488 nm, Fluorescence 525 nm).
  • FIGS. 7 and 8 show The results of exposure of RFR-Alexa488 and TAT-Alexa488 to CHO-K1 cells and CHO-A745 cells.
  • FIG. 7 shows the results of exposure to TAT-Alexa488, and
  • FIG. 8 shows RFR-Alexa488. Both values assume that the amount of CHO-K1 cells taken up is 100%. From FIGS. 7 and 8, when TAT-Alexa488 was exposed, the uptake amount decreased in CHO-A745 cells, whereas when RFR-Alexa488 was exposed, the uptake amount did not change even in CHO-A745 cells. all right. From this, it was shown that the sugar chain on the cell surface has no effect on the intracellular translocation of the RFR peptide.
  • QD quantum dot
  • Qdot registered trademark
  • QD-SA Q10143MP, Thermo Fisher Scientific
  • biotin-RFR B-RFR in which biotin (manufactured by Sigma-Aldrich) was bound to an RFR peptide was prepared as a pair of streptavidin.
  • B-RFR consists of 3 equivalents of (+)-Biotin-N-hydroxysuccinimide ester and 6 equivalents of N- for a peptide resin having an RFR sequence obtained by synthesizing biotin and RFR peptide by the Fmoc solid phase method. Obtained by reacting Methylmorpholine in DMF. After the reaction, B-RFR was purified using the above peptide purification method. The purified B-RFR was recovered using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer to confirm the RFR peptide bound to biotin.
  • the above-mentioned QD-SA-B-RFR-containing medium was added to the washed cells and cultured (medium volume 100 ⁇ L, 37 ° C., 5% CO 2 ). After culturing, 80 nM Hoechst 33342 was added, and the cells were stained at 37 ° C. for 15 minutes. After staining, the cells were washed twice with DMEM medium containing 10% FBS, and then the cells were observed with a confocal laser scanning microscope. For comparison, 5 nM QD-SA was suspended in DMEM medium containing 10% FBS instead of QD-SA-B-RFR, or 5 nM QD-SA and 20 nM RFR peptide (unbound biotin) were used. A medium suspended in DMEM medium containing 10% FBS was prepared, the same operation as above was performed, and the cells were observed with a confocal laser microscope.
  • FIGS. 9A-9C show the observation results.
  • FIG. 9A shows the result of adding QD-SA-B-RFR
  • FIG. 9B shows 5 nM QD-SA and 20 nM RFR peptide (without biotin) when only QD-SA was added.
  • the result of adding the mixed solution is shown.
  • the dotted lines in FIGS. 9A-9C show the outline of the cell nucleus.
  • the arrow portion in FIG. 9A is the fluorescence derived from the quantum dots, and it can be seen that the quantum dots are transferred to the cells.
  • fluorescence derived from quantum dots was not observed in the cells, indicating that the RFR peptide transferred the quantum dots into the cells.
  • Example 5 Intracellular introduction test of shepherdine peptide
  • Survivin is a gene related to apoptosis that is highly expressed in cancer cells, and the survivin protein suppresses apoptosis.
  • the peptide represented by KHSSGCAFL (SEQ ID NO: 4) in the survivin sequence is called a shepherdin peptide, and it induces cell death by inhibiting the interaction between survivin and HSP90 (Heat Shock Protein). It is expected to be used as an anticancer agent.
  • HSP90 Heat Shock Protein
  • RFR-shepherdin peptide (RFRYIQNLVTKNFPNKFGGKHSSGCAFL: SEQ ID NO: 5) consisting of a sequence linked to the RFR sequence was synthesized from the amino acid sequence of the shepherdine peptide represented by SEQ ID NO: 4. The above peptide synthesis method was used as the synthesis method.
  • a TAT-shepherdin peptide (GRKKRRQRRRPPQGGKHSSGCAFL: SEQ ID NO: 6) consisting of a sequence in which the amino acid sequence of the shepherdin peptide represented by SEQ ID NO: 4 is bound to the TAT sequence instead of the RFR sequence was synthesized.
  • This RFR-shepherdin was suspended in DMEM medium containing 10% FBS to prepare fluorescently labeled peptide-containing medium 9 (2 ⁇ M RFR-shepherdin, DMEM medium containing 10% FBS).
  • TAT-shepherdin was suspended in DMEM medium containing 10% FBS to prepare a fluorescently labeled peptide-containing medium 10 (2 ⁇ M TAT-shepherdin, DMEM medium containing 10% FBS).
  • A431 cells of epidermoid carcinoma were used as an exposure target for the RFR-shepherdin and TAT-shepherdin peptides.
  • A431 cells (12,000 cells) were added to 96-well microplates containing 100 ⁇ l of MEM medium containing 10% FBS and cultured for 24 hours. After removing the culture solution, fluorescently labeled peptide-containing medium 9 or fluorescently labeled peptide-containing medium 10 was added to A431 cells, respectively, and the cells were cultured for 48 hours (37 ° C.). After culturing, the cells were washed with MEM medium containing 10% FBS, and 100 ⁇ L of MEM medium containing 10% FBS was added.
  • FIGS. 10A to 10C The observation results of A431 cells are shown in FIGS. 10A to 10C.
  • the measurement result of the survival rate is shown in FIG. FIG. 10A shows the control
  • FIG. 10B shows the result of exposure to TAT-shepherdin
  • FIG. 10C shows the result of exposure to RFR-shepherdin.
  • FIG. 10C a large number of dead cells not found in FIGS. 10A and 10B were observed, such as the cells indicated by the arrows in FIG. 10C.
  • FIG. 11 it was shown that the viability of A431 cells was significantly reduced when RFR-shepherdin was exposed, and when RFR-shepherdin was exposed, RFR-shepherdin was contained in A431 cells. It was taken up and showed that cell death was induced.
  • Example 6 Transfection with RFR peptide
  • a cationic lipid Lipofectamine LTX (manufactured by Thermo Fisher Scientific) was used for transfection. HeLa cells were used as cells.
  • pEGFP-N1 manufactured by Clontech
  • 20 ng of pEGFP-N1 was added to 2 ⁇ L of Lipofectamine LTX, and DMEM medium was added to prepare a transfection medium so that the final volume was 20 ⁇ l, and the mixture was left at 25 ° C. for 20 minutes.
  • HeLa cells (2 x 10 5 cells) were added to 2 ml of DMEM medium containing 10% FBS in a 35 mm glass-based dish and cultured for 24 hours (37 ° C., 5% CO 2 ). After removing the culture medium, cells were cultured in the culture medium containing 20 ⁇ l of transfection medium in 80 ⁇ l of DMEM medium containing 10% FBS. At that time, the RFR peptide prepared in Example 1 was added so as to have a final concentration of 10 ⁇ M, and the cells were cultured at 37 ° C. for 24 hours. After culturing, 80 nM Hoechst 33342 was added, and the cells were stained at 37 ° C. for 15 minutes. After staining, the cells were washed twice with DMEM medium containing 10% FBS, and then the cells were observed with a confocal laser scanning microscope.
  • FIGS. 12A and 12B The results of transfection are shown in FIGS. 12A and 12B.
  • FIG. 12A shows the result of transfection without containing the RFR peptide
  • FIG. 12B shows the result of transfection with the addition of the RFR peptide.
  • Arrows in FIG. 12B indicate transfected cells. From FIGS. 12A and 12B, cells that were significantly transfected were obtained by transfection with the addition of RFR peptide. In this transfection test, the gene amount is smaller than that in the normal transfection test, so that the normal transfection is unlikely to occur. From this, it was shown that the addition of the RFR peptide promoted gene transfer into cells.
  • Example 7 (Base substitution of RFR peptide) Mutations were made to replace the amino acid sequence of the RFR peptide, and the effect of the mutant peptide on the intracellular uptake was measured.
  • the modified fluorescently labeled peptide shown in Table 1 below was prepared using the same method as the method for producing the fluorescently labeled peptide.
  • Alexa 488 indicates a fluorescent dye.
  • each modified fluorescently labeled peptide was exposed to Hela cells in the same manner as in Example 1, and a flow cytometer was used. The amount of cell fluorescence of 10,000 living cells was measured using the cell.
  • FIGS. 13A to 13C The measurement results of the cell fluorescence amount are shown in FIGS. 13A to 13C.
  • the results using fluorescently labeled peptide-containing medium 2 that is, unmodified RFR-Alexa 488) are also shown. From the results of FIGS. 13A to 13C, it was shown that each modified peptide also has the ability to translocate into cells.
  • Example 8 (Verification of water solubility of RFR peptide) The water solubility of the RFR peptide was verified.
  • the above-mentioned fluorescently labeled peptide was synthesized by synthesizing a polypeptide consisting of each of the RFR sequence and the TAT sequence using the above-mentioned peptide synthesis method, and Alexa750 was used instead of Alexa488 for these synthetic peptides. Alexa750 was added using the same method as in the above synthesis method, and Alexa750-labeled RFR (RFR-Alexa750) and Alexa750-labeled TAT (TAT-Alexa750) were synthesized and used.
  • FIG. 14A After lyophilization, 500 ⁇ l of pure water was added to each centrifuge tube, and the mixture was stirred for 30 seconds using a vortex mixer. The result of observing the aqueous solution after stirring is shown in FIG. 14B. From FIG.
  • TAT-Alexa750 adsorbed a large amount on the wall surface of the centrifuge tube and had low solubility in water
  • RFR-Alexa750 was dissolved in water without adsorbing on the wall surface of the centrifuge tube. From this, it was shown that the RFR peptide has high solubility in water.

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Abstract

La présente invention concerne un peptide pénétrant les cellules. La présente invention concerne : un complexe contenant le peptide pénétrant les cellules et une molécule cible ; et un procédé de production du complexe. La présente invention concerne : un réactif contenant le peptide pénétrant les cellules ou le complexe ; une préparation pharmaceutique contenant le complexe ; un procédé pour améliorer la capacité de pénétration cellulaire d'une molécule cible ; un procédé de transfection pour une molécule d'acide nucléique ; et un procédé pour introduire une molécule cible à l'intérieur d'une cellule.
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Citations (4)

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WO2011010337A1 (fr) * 2009-07-21 2011-01-27 Enea - Ente Per Le Nuove Technologie, L'energia E L'ambiente Vaccins à base de chimère génétique d’antigènes viraux et/ou tumoraux et de protéines végétales
WO2016052442A1 (fr) * 2014-09-29 2016-04-07 国立大学法人京都大学 Peptide de transport vers le cytoplasme
WO2016076347A1 (fr) * 2014-11-13 2016-05-19 東亞合成株式会社 Procédé d'introduction de substance exogène dans une cellule, et matériau utilisé dans ledit procédé
JP2016523088A (ja) * 2013-06-26 2016-08-08 フィロジカ リミテッドＰｈｙｌｏｇｉｃａ Ｌｉｍｉｔｅｄ ペプチドの細胞輸送をモニタリングする方法

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WO2011010337A1 (fr) * 2009-07-21 2011-01-27 Enea - Ente Per Le Nuove Technologie, L'energia E L'ambiente Vaccins à base de chimère génétique d’antigènes viraux et/ou tumoraux et de protéines végétales
JP2016523088A (ja) * 2013-06-26 2016-08-08 フィロジカ リミテッドＰｈｙｌｏｇｉｃａ Ｌｉｍｉｔｅｄ ペプチドの細胞輸送をモニタリングする方法
WO2016052442A1 (fr) * 2014-09-29 2016-04-07 国立大学法人京都大学 Peptide de transport vers le cytoplasme
WO2016076347A1 (fr) * 2014-11-13 2016-05-19 東亞合成株式会社 Procédé d'introduction de substance exogène dans une cellule, et matériau utilisé dans ledit procédé

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FUTAKI S. ET AL.: "Cell-Surface Interactions on Arginine-Rich Cell - Penetrating Peptides Allow for Multiplex Modes of Internalization", ACC. CHEM. RES., vol. 50, no. 10, 17 October 2017 (2017-10-17), pages 2449 - 2456, XP055796955 *
KOSUI, MOMOKO ET AL: "Examination of internalization sequence of saporin toxin", ABSTRACTS OF THE 92ND ANNAL MEETING OF THE JAPANESE BIOCHEMICAL SOCIETY, 4 September 2019 (2019-09-04), pages 684 *
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