WO2021040022A1 - Cell-penetrating peptide and use thereof - Google Patents

Abstract

The present invention relates to a cell-penetrating peptide. The present invention relates to: a complex containing the cell-penetrating peptide and a target molecule; and a method for producing the complex. The present invention relates to: a reagent containing the cell-penetrating peptide or the complex; a pharmaceutical preparation containing the complex; a method for improving the cell-penetrating capability of a target molecule; a transfection method for a nucleic acid molecule; and a method for introducing a target molecule to inside a cell.

Description

Membrane-permeable peptides and their uses

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. Furthermore, 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.

As one of the DDS methods, 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.

Endocytosis and direct membrane permeation have been proposed as the mechanism of intracellular translocation of membrane-permeable peptides, and endocytosis or direct membrane permeation is proposed for intracellular delivery using actual membrane-permeable peptides. It is believed that this is done by a route through or a combination thereof. 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). Polypeptides that are arginine residues), penetratin, which is a basic helix peptide derived from the antennapedia homeodomain protein of Drosophila, and the like. Attention has been paid to the basicity of the sequences of these peptides, such as oligoarginine. It is considered that the peptide is introduced into the cell by attaching a membrane-permeable peptide having a positive charge to the negative charge on the cell membrane surface (for example, glycoprotein, lipid, etc.) (Patent Document 1). ). However, conventional 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.

Japanese Unexamined Patent Publication No. 2013-100273

There is a demand for a membrane-permeable peptide with reduced non-specific adsorption compared to conventional membrane-permeable peptides.

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.

That is, 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. Furthermore, 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. Furthermore, the present invention provides a reagent containing the above-mentioned membrane-permeable peptide or the above-mentioned complex. Furthermore, 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.

According to the present invention, a membrane-permeable peptide with reduced non-specific adsorption is provided. It can be used for DDS of drugs and research reagents.

As a model of a drug to be introduced into cells, it is an image showing the cell fluorescence signal of HeLa cells exposed to fluorescently labeled peptide-containing media 1 and 3 containing RFR peptide labeled with the fluorescent dye Alexa Fluor 488. It is a graph which shows the cell fluorescence amount of the HeLa cell which exposed the fluorescently labeled peptide containing culture medium 1 and 3. 3 is an image showing a cell fluorescence signal of HeLa cells exposed to fluorescently labeled peptide-containing media 2 and 4. It is a graph which shows the cell fluorescence amount of the HeLa cell which exposed the fluorescently labeled peptide containing mediums 2 and 4. 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. It is an observation image by a confocal laser scanning microscope of HeLa cells to which only QD-SA was added. It is an observation image by a confocal laser scanning microscope of HeLa cells to which a solution which mixed QD-SA and a biotin-unbound RFR peptide was added. It is a cell image of A431 cells exposed to a control sample. It is a cell image of A431 cells exposed to TAT-shepherdin. It is a cell image of A431 cells exposed to RFR-shepherdin. It is a graph which shows the cell viability of A431 cells exposed to a shepherdine peptide. It is a cell image after a transfection test without using an RFR peptide. It is a cell image after a transfection test using an RFR peptide. It is a graph which shows the cell fluorescence amount of the HeLa cell using the modified fluorescently labeled peptide. It is a graph which shows the cell fluorescence amount of the HeLa cell using the modified fluorescently labeled peptide. It is a graph which shows the cell fluorescence amount of the HeLa cell using the modified fluorescently labeled peptide. It is an image of TAT-Alexa750 or RFR-Alexa750 in the centrifuge tube after lyophilization. It is an image of TAT-Alexa750 or RFR-Alexa750 in the centrifuge tube after resuspension.

In this specification, various 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.

In the amino acid sequence represented by SEQ ID NO: 1, 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. Specifically, 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. It may be selected from substitutions that make glutamine residues alanine residues or substitutions that make the 7, 12 and 15th asparagine residues alanine residues. Here, 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.). ).

In another embodiment, 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. Good. 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. Here, 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).
Further, 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.

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 μM 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 μM or less. Has. Furthermore, 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. For example, the final concentration of the membrane-permeable peptide in the solution or medium is 1M, 500μM, 100μM, 50μM, 20μM, 10μM, 9μM, 8μM, 7μM, 6μM, 5μM, 4μM, 3μM, 2μM, 1μM, 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. For example, a peptide solid phase synthesis method such as Fmoc synthesis method or Boc synthesis method can be used.
Alternatively, 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. For example, 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.
In the present invention, 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. Examples of 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. In this case, by modifying the end of the membrane-permeable peptide with biotins and binding avidins to the target molecule, the membrane-permeable peptide and the target molecule can be indirectly bound. 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. By binding 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. Further, if 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.
As 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. As 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. If 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. As 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.
In addition to the membrane-permeable peptide or the above-mentioned complex, 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.

As such an additive, it can be appropriately selected from pharmaceutically acceptable additives. 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.), disintegrants. (Starches, crystalline cellulose, croscarmellose sodium, crospovidone, etc.), non-aqueous excipients (almond 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. Further, 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. Specifically, the final concentration of the membrane-permeable peptide in the solution is 1M, 500μM, 100μM, 50μM, 20μM, 10μM, 9μM, 8μM, 7μM, 6μM, 5μM, 4μM, 3μM, 2μM, 1μM, 500nM, 400. , 300nM, 200nM, 100nM, 50nM, 10nM, 5nM, 1nM can be added. When the reagent or the pharmaceutical preparation is in the solid form, 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. Specifically, 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. , 50 parts by weight, 40 parts by weight, 30 parts by weight, 20 parts by weight, 15 parts by weight, 10 parts by weight, 9 parts by weight, 8 parts by weight, 7 parts by weight, 6 parts by weight, 5 parts by weight, 4 parts by weight, 3 It is possible to add parts by weight, 2 parts by weight, 1 part by weight, 0.5 parts by weight, 0.4 parts by weight, 0.3 parts by weight, 0.2 parts by weight, and 0.1 parts by weight.

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. By binding the target molecule to the membrane-permeable peptide, the membrane permeability of the target molecule is improved, and substances that were originally impervious to or difficult to permeate the cell membrane can be transported intracellularly, especially to the cytosol. it can.

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. Therefore, 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. When the nucleic acid molecule is a protein expression vector, for example, 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. As such a transfection reagent, for example, it can be used in combination with a transfection reagent by the lipofection method, a transfection reagent by the calcium phosphate method, or the like. In a preferred embodiment, the transfection method comprises mixing a membrane-permeable peptide, a nucleic acid molecule, and a transfection reagent to form a complex. When 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.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples.

(Peptide synthesis)
All peptides used in the present invention were synthesized by the Fmoc (Fluorenyl-MethOxy-Carbonyl) solid phase method. The synthetic operation conforms to the Fmoc amino acid synthesis method generally used by those skilled in the art.
As an automatic peptide synthesizer, PSSM-8 manufactured by Shimadzu Corporation, a resin for peptide synthesis (TGS-RAM, Shimadzu Corporation) as a solid phase, and an Fmoc amino acid (Peptide Research Institute) in which a protecting group was added to the amino acid to be condensed were prepared. Add Fmoc amino acid to the resin and add 1-hydroxybenzotriazole (HOBt) / 2- (1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) / N , Fmoc amino acid condensation reaction using N-diisopropylethylamine (DIEA) and Fmoc deprotection reaction (30% piperidine) were carried out. By repeating this operation, a peptide chain elongation reaction was carried out.
(Peptide purification)
After the extension reaction of the peptide chain of interest on the resin was completed, deprotection and separation of the peptide from the resin was performed using a solution of trifluoroacetic acid (TFA) -ethanedithiol (EDT) (95: 5) (20 ° C.). ,3 hours). The crude peptide excised from the resin was subjected to reverse phase high performance liquid chromatography (Chromaster, Hitachi High-Tech Science) set with a 5C18-AR-II Cosmosil column (Nacalai Tesque), and contained two solvents: 0.1% TFA. Purified using H _{2 O and CH 3} CN containing 0.1% TFA.
After lyophilization, the molecular weight of the target peptide was confirmed using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (Microflex, Bruker Daltonics).

(Synthesis of fluorescently labeled peptide)
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.

(Exposure experiment of fluorescently labeled peptide)
Using the above peptide synthesis method, a polypeptide consisting of the RFR sequence (RFRYIQNLVTKNFPNKF: SEQ ID NO: 1) derived from saporin toxin (SEQ ID NO: 2) and the TAT sequence derived from HIV (GRKKRRQRRRPPQ: SEQ ID NO: 3) was synthesized. Alexa 488 was added to these synthetic peptides using the above fluorescently labeled peptide synthesis method, and Alexa 488-labeled RFR (RFR-Alexa 488) and Alexa 488-labeled TAT (TAT-Alexa 488) were synthesized.
HeLa cells (2 × 10 ⁵ cells, 2 mL) 24 hours in DMEM medium 35 mm glass based dishes _{(37 ℃, 5% CO 2} ) after and the cells washed with DMEM medium (500 [mu] L, 3 times).
Medium containing 4 types of fluorescently labeled peptides:
DMEM medium containing 500nM RFR-Alexa488: Fluorescently labeled peptide-containing medium 1;
DMEM medium containing 500nM RFR-Alexa488, 10% 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 μL, 37 ° C., 5% CO ₂ ).
HeLa cells cultured in each fluorescently labeled peptide-containing medium are added to nuclear staining reagent (Hoechst 33342, 5 μg / mL, 200 μL) _{, incorporated into cells for 20 minutes (37 ° C, 5% CO 2} ), and then in DMEM medium. The cells were washed (500 μL, 3 times).
The cell fluorescence signal of these HeLa cells was observed using a confocal laser scanning microscope (FV1200, Olympus).

(Measurement of fluorescently labeled peptide-exposed cells by flow cytometer)
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 μL, 3 times). Fluorescently labeled peptide-containing media 1 to 4 were added to the washed cells and cultured for 30 minutes (medium volume 300 μL, 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 μL, 3 times). After washing, trypsin (0.1 g / L) treatment (200 μL, 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 μL) was added to disperse the cells. Centrifugation was performed again at 4 ° C. and 200 × g to remove the supernatant and add PBS (400 μL) twice.
The cell fluorescence of 10,000 living cells was measured using a flow cytometer (Guava easyCyte, Merck Millipore) for the washed cells.

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, and the right panel is a merged image of these. The lower right bar of each image represents the length of 20 μm 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. On the other hand, it was found that the non-specific adsorption of the dish on the glass surface was suppressed. On the other hand, it was found that TAT-Alexa488 had a larger background than RFR-Alexa488 and was non-specifically adsorbed on the glass surface of the dish. From FIG. 2, it was found that RFR-Alexa488 showed a stronger fluorescence amount than TAT-Alexa488, and RFR-Alexa488 was taken up by cells more often than TAT-Alexa488.
Next, the results of measuring the cell fluorescence signal and the amount of cell fluorescence of the fluorescently labeled peptide-containing media 2 and 4 are shown in FIGS. 3 (confocal laser scanning microscope observation) and FIG. 4 (flow cytometer measurement). Similar to FIGS. 1 and 2, it was found that RFR-Alexa488 was taken up by cells more often than TAT-Alexa488 in FIGS. 3 and 4, and RFR-Alexa488 was taken up by cells more often even in the presence of FBS. I found out.

Evaluation of intracellular translocation of fluorescently labeled peptide In the above-mentioned exposure of fluorescently labeled peptide to HeLa cells, DMEM containing fluorescently labeled peptide-containing medium 5 (2 μM RFR-Alexa488, 10% FBS) instead of fluorescently labeled peptide-containing mediums 2 and 4. The same experiment was performed except that the medium) and the medium 6 containing fluorescently labeled peptide (2 μM TAT-Alexa488, DMEM medium containing 10% FBS) were used, and the cell fluorescence signal was observed using a confocal laser microscope.

The evaluation of intracellular migration of the fluorescently labeled peptide is shown in FIG. It was found that the fluorescence of RFR-Alexa488 was strong in the cytosol region of the cell, and RFR-Alexa488 was taken up into the cell and transported to the cytosol region in large quantities.

(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 μL) were cultured in 96 well microplates (IWAKI) for 24 hours (37 ° C, 5% CO ₂ ), then cell-washed in DMEM medium (50 μL, 3 times) and fluorescently labeled. Peptide-containing medium 1 or 3 was added and cultured for 30 minutes (medium volume 50 μL, 37 ° C., 5% CO ₂ ).
Then, WST-1 reagent (10 μL) 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. As a control, 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.

From the above results, it was found that 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. In addition, 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. Here, the sugar chain dependence of the RFR peptide of the present invention was examined.

As the peptides, the above RFR-Alexa488 and TAT-Alexa488 were used. As a medium to be exposed to cells, a fluorescently labeled peptide-containing medium 7 in which RFR-Alexa488 is suspended so that the final concentration is 500 nM in F-12 medium (manufactured by Gibco), and 500 nM in F-12 medium. 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 μL, 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).

The results of exposure of RFR-Alexa488 and TAT-Alexa488 to CHO-K1 cells and CHO-A745 cells are shown in FIGS. 7 and 8. 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.

(Example 4)
Intracellular introduction test of quantum dots using RFR peptide Quantum dots are expected to play a role as a biosensor due to their strong fluorescence intensity, but they are known to have low transfer efficiency into cells. Here, we evaluated the efficiency of intracellular introduction of quantum dots by RFR peptides.

As a quantum dot (hereinafter, also referred to as QD) sample, Qdot (registered trademark) 525-Streptavidin Conjugate (QD-SA: Q10143MP, Thermo Fisher Scientific) in which streptavidin was bound to QD was used.
In addition, 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.

Mix 0.5 μL of 5 nM QD-SA and B-RFR solution in 2 μL of PBS so that the final concentration of B-RFR is 20 nM, and leave it for 30 minutes via streptavidin and biotin. QD and RFR were combined (QD-SA-B-RFR). This QD-SA-B-RFR was suspended in DMEM medium containing 10% FBS (QD-SA-B-RFR-containing medium).
HeLa cells (1 × 10 ⁵ cells) were cultured in DMEM medium containing 10% FBS in a 35 mm glass-based dish for 48 hours (37 ° C., 5% CO ₂ ), and then the cells were washed with DMEM medium. The above-mentioned QD-SA-B-RFR-containing medium was added to the washed cells and cultured (medium volume 100 μL, 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.

The observation results are shown in FIGS. 9A-9C, respectively. FIG. 9A shows the result of adding QD-SA-B-RFR, and 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. In FIGS. 9B and 9C, 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. Here, we examined the intracellular introduction of shepherdine peptide using RFR peptide.

An 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. Similarly, 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 μM RFR-shepherdin, DMEM medium containing 10% FBS). In addition, TAT-shepherdin was suspended in DMEM medium containing 10% FBS to prepare a fluorescently labeled peptide-containing medium 10 (2 μM TAT-shepherdin, DMEM medium containing 10% FBS).

As an exposure target for the RFR-shepherdin and TAT-shepherdin peptides, A431 cells of epidermoid carcinoma (ATCC) were used.
A431 cells (12,000 cells) were added to 96-well microplates containing 100 μl 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 μL of MEM medium containing 10% FBS was added. Some cells were observed using a culture microscope (CKX31: Olympus). Then, 10 μl of WST-1 reagent was added and cultured in the same manner as in Example 2, and the viability of A431 cells was measured by measuring the absorbance. For comparison, the same operation was performed using MEM medium (control) containing 10% FBS or MEM medium containing 10% FBS and Shepherdine peptide instead of the fluorescently labeled peptide-containing medium 9 or the fluorescently labeled peptide-containing medium 10. , A431 cells viability was measured.

The observation results of A431 cells are shown in FIGS. 10A to 10C. In addition, 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, and FIG. 10C shows the result of exposure to RFR-shepherdin. In 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. Further, as shown in 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)
Cell transfection tests were performed using RFR peptides. A cationic lipid, Lipofectamine LTX (manufactured by Thermo Fisher Scientific), was used for transfection. HeLa cells were used as cells. As the introduced gene, pEGFP-N1 (manufactured by Clontech) was used.
20 ng of pEGFP-N1 was added to 2 μL of Lipofectamine LTX, and DMEM medium was added to prepare a transfection medium so that the final volume was 20 μl, 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 μl of transfection medium in 80 μl 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 μM, 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.

The results of transfection are shown in FIGS. 12A and 12B. FIG. 12A shows the result of transfection without containing the RFR peptide, and 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. In the table, Alexa 488 indicates a fluorescent dye.

Except that each of the above-mentioned modified fluorescently labeled peptides was used instead of RFR-Alexa488 of the fluorescently labeled peptide-containing medium 2, 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.

The measurement results of the cell fluorescence amount are shown in FIGS. 13A to 13C. For comparison, 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. In the experiment, 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.

Aqueous solutions of RFR-Alexa750 and TAT-Alexa750 were added to each 15 ml volume polypropylene centrifuge tube (IWAKI). These were subjected to freeze-drying to remove the solvent. The result of observing each centrifuge tube from which the solvent was removed is shown in FIG. 14A. After lyophilization, 500 μl 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. 14B, TAT-Alexa750 adsorbed a large amount on the wall surface of the centrifuge tube and had low solubility in water, whereas 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.

Claims (16)

A membrane-permeable peptide 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. The membrane-permeable peptide according to claim 1, wherein an additional sequence consisting of 1 or more and 35 or less amino acid residues is added to at least one of the N-terminal and C-terminal of the membrane-permeable peptide. In the amino acid sequence represented by SEQ ID NO: 1, the deleted or substituted amino acid sequence is deleted or substituted with 1 or more and 10 or less amino acid residues other than the lysine residue, arginine residue and phenylalanine residue. The membrane-permeable peptide according to claim 1 or 2, which is an amino acid sequence. The membrane-permeable peptide according to any one of claims 1 to 3, wherein the amino acid residue to be added to the membrane-permeable peptide is selected from a lysine residue, an arginine residue, and a phenylalanine residue. In the membrane-permeable peptide, the amino acid residues to be substituted are the 1st arginine residue, the 3rd arginine residue, the 11th lysine residue, and the 16th lysine in the amino acid sequence represented by SEQ ID NO: 1. Residues, 2nd phenylalanine residue, 13th phenylalanine residue, 17th phenylalanine residue, 4-10, 12, 14 and 15th amino acid residues, 6th glutamine residue, 7, 12 and The membrane-permeable peptide according to any one of claims 1, 2 or 4, which is selected from at least the 15th asparagine residue. In the membrane-permeable peptide, the amino acid residue to be substituted is a substitution in which the first arginine residue of the amino acid sequence represented by SEQ ID NO: 1 is an alanine residue, and the third arginine residue is an alanine residue. Substitution to make the 11th lysine residue an alanine residue, substitution to make the 16th lysine residue an alanine residue, substitution to make the 2nd phenylalanine residue an alanine residue, 13th phenylalanine residue Substitution with group as alanine residue, substitution with 17th phenylalanine residue as alanine residue, substitution with 4-10, 12, 14 and 15 amino acid residues as alanine residue, 6th glutamine residue The membrane according to any one of claims 1, 2, 4 or 5, which is selected at least from a substitution in which the group is an alanine residue or a substitution in which the asparagine residues at positions 7, 12 and 15 are alanine residues. Permeable peptide. A membrane-permeable peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 7 to SEQ ID NO: 16, 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 according to any one of claims 1 to 5 or 7, wherein the substitution is a conservative substitution. A complex containing the membrane-permeable peptide according to any one of claims 1 to 8 and a target molecule. The complex according to claim 9, wherein the target molecule is a pharmaceutical compound. The method for producing a complex according to claim 9 or 10, which comprises binding the membrane-permeable peptide according to any one of claims 1 to 8 to a target molecule. A reagent containing the membrane-permeable peptide according to any one of claims 1 to 8 or the complex according to claim 9 or 10. A pharmaceutical preparation containing the complex according to claim 9 or 10. A method for improving the membrane permeability of a target molecule, which comprises binding the membrane-permeable peptide according to any one of claims 1 to 8 to a target molecule. A method for transfecting a nucleic acid molecule, which comprises mixing the membrane-permeable peptide according to any one of claims 1 to 8 with a nucleic acid molecule. A membrane consisting of 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 sequence in which 1 to 10 amino acid residues are deleted, substituted or added in the amino acid sequence. A method of introducing a target molecule into a cell using a permeable peptide.

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