Intracellular protein delivery agent and intracellular protein introduction method

Abstract

To provide a protein delivery agent and an intracellular protein introduction method capable of efficiently transporting proteins into cells and / or nuclei without showing cytotoxicity.SOLUTION: An intracellular protein delivery agent is a complex formed by at least cationic carrier molecules and anionic carrier molecules, which are bonded to each other by at least ionic bonds and hydrophobic interactions. Preferably, the complex is formed by cationic carrier molecules, anionic carrier molecules, and a protein, which are bonded to each other by at least ionic bonds and hydrophobic interactions. An intracellular protein introduction method uses the intracellular protein delivery agent. Here, the intracellular protein delivery agent may be an oligomer or polymer in which the cationic carrier molecule has a cationic group as a repeating unit.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a protein delivery agent and a protein introduction method for introducing a protein into a cytoplasm. [Background technology]

[0002] Protein transfection technology is a technique for directly introducing various proteins such as antibodies into cells, and compared to the conventional method of inducing expression of a target protein by gene introduction such as a plasmid, it has been actively researched in recent years as a new method that avoids the potential risks and problems of low expression efficiency associated with genetic recombination. At present, it is an extremely difficult task to transport hydrophilic and high molecular weight substances such as proteins through highly hydrophobic cell membranes and into the cytosol of living cells while maintaining their activity.

[0003] So far, various methods have been developed for transporting relatively low molecular weight compounds into living cells. For example, in Non-Patent Document 1, a basic peptide containing multiple consecutive arginine residues has been used as a carrier molecule to transport various low molecular weight compounds, nucleic acids, proteins, etc. into cells. However, this method has a problem that the introduction rate into cells decreases as the molecular weight of the substance to be transported into cells increases. Although the introduction efficiency can be improved to some extent by increasing the repeating units of arginine residues and increasing the length of the peptide chain, there is a problem that the interaction with cell membranes and proteins becomes stronger and cytotoxicity becomes significant. When the transport molecule is a low molecular weight, peptides and low molecules having arginine residues may directly permeate the lipid membrane, but there is a problem that they cannot pass through the lipid membrane if the lipid membrane is a zwitterionic phosphocholine lipid membrane.

[0004] In order to increase the efficiency of transferring substances into cells using the above-mentioned basic peptides, the effect of introducing pyrene butyrate, which functions as an activator molecule, is shown in Non-Patent Document 2. In this case, it has been shown that the efficiency of introducing various bioactive molecules using a peptide having an arginine residue after previously introducing pyrene butyrate to the cell surface is improved, but since the peptide and pyrene butyrate aggregate when mixed, they must be introduced to the cell surface separately, and further, if there is no bond between the molecule to be transported and these carrier molecules and activator molecules, interactions occur with various proteins in the environment including the cells, making it difficult to transport the target molecule.

[0005] Unlike the direct permeation of the lipid membrane by carrier molecules and transport molecules, uptake into cells by endocytosis also occurs, but in this case, there are problems with the contents of the substances encapsulated in endosomes being decomposed by lysosome fusion, and with the contents not being able to escape from the endosome, making it difficult to utilize the activity of the transported substances. Furthermore, when considering in vivo use, peptides containing arginine residues strongly interact with various proteins in serum such as albumin, which impedes the transport of substances, making the use of such basic peptides difficult, especially when considering clinical applications.

[0006] Non-Patent Document 3 reports that boron cluster anions, spherical anionic molecules formed by the assembly of 12 boron and bromine atoms, can be used as carrier molecules to transport various low- to medium-molecular-weight compounds into the cytoplasm through the cell membrane. The boron cluster anion has the property of being able to pass through the cell membrane quickly without destroying it itself and be efficiently introduced into the cytoplasm. In addition, the boron cluster anion is characterized by its strong chaotropic properties, which allow it to bind to and stabilize various molecules. It is described that due to these properties, complexes of boron clusters with various compounds can be suitably used to pass through the cell membrane and deliver compounds into the cytoplasm. However, in this case, the molecules that can be delivered into the cytoplasm are limited to compounds with a molecular weight range of at most 4500 or less, and proteins such as albumin cannot be transported into the cell.

[0007] Patent Document 1 discloses a method of introducing a high molecular weight hydrophilic polymer into a cell by macropinocytosis by encapsulating a hydrophilic polymer such as a protein or a nucleic acid inside a liposome having a peptide on the surface thereof that contains multiple consecutive arginine residues. Macropinocytosis is also included in the mechanism of taking up a substance into a cell by endocytosis in a broad sense, and in this case, the lysosome does not fuse with the macropinosome, so that the degradation of the contents can be avoided. However, even in this case, there is a problem that it is difficult to release the contents from the macropinosome membrane.

[0008] Patent Document 2 discloses a method in which various proteins are transported into cells via endocytosis using an improved peptide obtained by modifying the hemolytic peptide M-lycotoxin derived from spider venom, and the delivered protein is released into the cytoplasm while retaining its activity by selectively destabilizing the endosomal membrane in the cytoplasm with the action of the peptide. There is concern that the peptide basically contains an element that destabilizes the cell membrane.

[0009] Non-Patent Document 4 discloses a method for introducing proteins such as lectins into cells using self-assembled cationic core-shell nanoparticles made of a copolymer having cationic and amphiphilic repeating units. In this method, delivery is difficult because the nanoparticles and cationic proteins are unlikely to bind to each other.

[0010] Non-Patent Document 5 reports that the method of Patent Document 2 was insufficient as an intracellular delivery agent for antibody proteins, but instead, "ProteoCarry" was used to efficiently deliver the antibody protein into cells and express its function. The structure of ProteoCarry has not been disclosed, and there is a problem that there is little knowledge about its adverse effects on the body and its safety. Furthermore, there is a concern that the transfection efficiency is not necessarily good due to its weak binding to the protein to be introduced.

[0011] In the transport of the various proteins mentioned above into cells, except when liposomes are used, the binding between the protein to be transported and peptides or other molecules that function as carriers is weak, which poses the problem that the pharmacokinetics of the carrier molecule and the protein to be transported differ when used in vivo. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication Pamphlet No. WO2005 / 032593 [Patent Document 2] International Publication Pamphlet No. WO2016 / 052442 [Non-patent literature]

[0013] [Non-Patent Document 1] Futaki, S. et al. Intracellular delivery technology using membrane-permeable basic peptides: its molecular mechanism and applications. Seibutsubushi, 50(3), 137-140 (2010). [Non-Patent Document 2] S. Katayama et al., “Effects of pyrenebutyrate on the translocation of arginine-rich cell-penetrating peptides through artificial membranes: Recruiting peptides to the membranes, dissipating liquid-ordered phases, and inducing curvature” Biochimica et Biophysica Acta (BBA)-Biomembranes, 1828(9), 2134-2142 (2013). [Non-Patent Document 3] A. Barba-Bon et al., “Boron clusters as broadband membrane carriers” Nature, 603(7902), 637-642 (2022). [Non-Patent Document 4] Ashlynn LZ Lee et al., “Efficient intracellular delivery of functional proteins using cationic polymer core / shell nanoparticles” Biomaterials, 29(9), 1224-1232 (2008). [Non-Patent Document 5] Y. Nakazato et al., “Protein Delivery to Insect Epithelial Cells In Vivo: Potential Application to Functional Molecular Analysis of Proteins in Butterfly Wing Development” BioTech, 12(2), 28 (2023). Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention aims to provide a carrier molecule that has the function of efficiently transporting proteins into cells, and to provide a protein delivery agent and an intracellular protein introduction method that are non-cytotoxic and can efficiently transport proteins into cells and / or the nucleus. [Means for solving the problem]

[0015] The intracellular protein delivery agent of the present invention is a complex consisting of a cationic carrier molecule, an anionic carrier molecule, and a protein, which are bound to each other through at least ionic bonds and hydrophobic interactions.

[0016] The intracellular protein delivery agent of the present invention may be such that the cationic carrier molecule is an oligomer or polymer having a cationic group in the repeating unit. Alternatively, in the intracellular protein delivery agent of the present invention, the cationic carrier molecule is preferably hexadimethrine bromide (HDB, trade name: Polybrene). Furthermore, polyethylenimine (PEI) may be added as the cationic carrier molecule. Alternatively, it is also preferable to use protamine as the cationic carrier molecule. In the intracellular protein delivery agent of the present invention, the protein may be an antibody, an enzyme, or a nucleic acid-protein complex (RNP: ribonucleoprotein) used in CRISPR / Cas9.

[0017] The intracellular protein delivery agent of the present invention may be such that the anionic carrier molecule is a boron cluster salt represented by the following general formula 1. In the following general formula 1, M represents a cation selected from lithium, sodium, potassium, and cesium, B represents a boron atom, X represents hydrogen or a halogen atom (F to I), and n represents 10 or 12.

[0018] (chemical 1) M 2+ (B n X n )2- (General formula 1)

[0019] The method for introducing a protein into an cell of the present invention comprises the following steps 1) to 4). 1) An intracellular protein delivery agent is prepared by mixing a solution in which a cationic carrier molecule is dissolved, a solution in which an anionic carrier molecule is dissolved, and a solution in which a protein is dissolved (first step). 2) Contacting the intracellular protein delivery agent with the cell surface (second step). 3) The intracellular protein delivery agent is introduced into the cytoplasm by endocytosis, encapsulated in the endosomal membrane (third step). 4) The intracellular protein delivery agent escapes the endosomal membrane and diffuses into the cytoplasm (fourth step).

[0020] The method for introducing an intracellular protein of the present invention may further comprise the step (5) of diffusing the intracellular protein delivery agent into the cell nucleus (fifth step). Effect of the Invention

[0021] According to the present invention, there is provided a carrier molecule having a function of efficiently transporting a protein into a cell, and further, there is an effect of providing a protein delivery agent and an intracellular protein introduction method that are capable of efficiently transporting a protein into a cell and / or nucleus without exhibiting cytotoxicity and capable of expressing its function in the cell. Furthermore, there is an effect that when the carrier molecule and the protein are integrated and stable in a complex, it is possible to match the pharmacokinetics of each of them. [Brief description of the drawings]

[0022] [Figure 1] A schematic diagram of a complex formed by binding both cationic and anionic carrier molecules to a protein, which has the function of transporting the protein into the cell. [Diagram 2] Fluorescence microscopy image showing delivery of green fluorescent protein into the cell nucleus [Diagram 3] Flow diagram of the method for introducing green fluorescent protein into cell nuclei [Figure 4] Fluorescence microscopy image showing intracellular delivery of secondary antibodies [Diagram 5] Fluorescence microscopy image (A) showing protein delivery into the cell nucleus in Example 3, and a graph (B) showing the delivery efficiency. [Figure 6] Fluorescence microscopy images showing protein delivery to the cytosol in Example 4. [Figure 7] Fluorescence microscopy images showing secondary antibody delivery to the cytosol in Example 4. [Figure 8] Fluorescence microscopy images showing protein delivery to the cytosol in Example 5. [Figure 9] Graph showing average particle size (DLS measurement results) based on scattering intensity distribution of complexes of Cs2B12Br12 with HDB and BSA DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention is characterized in that it uses a complex consisting of a cationic carrier molecule, an anionic carrier molecule, and a protein bound to each other by ionic bonds and / or hydrophobic interactions in order to transport various proteins into cells. The complex of the present invention is shown in FIG. 1.

[0024] The cationic carrier molecule has the function of adsorbing to the negatively charged phospholipid membrane surface and adhering the complex to the cell surface. Furthermore, the cationic carrier molecule is adsorbed to the surface of the protein to be transported by electrostatic interaction, so that when the complex is introduced into the body, the dynamics of the carrier molecule and the protein in the body can be matched until they are transported into the cell. This means that the anionic carrier molecule contained in the complex is bound to the cationic carrier molecule by electrostatic interaction and is also bound to the protein by interactions such as electrostatic interaction or hydrophobic interaction, which play an important role in matching the dynamics of the entire complex in vivo.

[0025] Although the interactions between these molecules are mutually reversible, the electrostatic interactions and hydrophobic interactions of each of them work synergistically, so that the complex is characterized by maintaining the integration of the complex in the interstitial fluid while suppressing the dissociation of each component molecule from the complex. Among the components of the complex, the anionic carrier molecule binds to both the cationic carrier molecule and the protein, and integrates them together, and has the function of permeating the lipid membrane, so that it is preferable that it has high hydrophobicity and good affinity with the lipid membrane. As an example of the anionic carrier molecule, for example, a boron cluster group or the like is most preferably used.

[0026] Cationic carrier molecules that can be preferably used in the complex of the present invention include cationic oligomers or polymers having a cationic group in the repeating unit. Here, the cationic group means a group that is cationic charged in an aqueous solution having a pH value between near neutral and near weak acidity, and specifically, it is preferable that the basic nitrogen atom is cationic charged by adding a proton. As cationic oligomers or polymers, those that strongly interact with lipid membranes such as oligolysine and oligoarginine that are formed by linking amino acids having amino groups in the side chains such as lysine, arginine, and histidine, basic polymers having primary amines in the side chains such as polyallylamine, polymers having secondary amines in the main chain such as polyethyleneimine, or other water-soluble cationic polymers having basic nitrogen atoms can be preferably used.

[0027] The cationic polymer may also be used in the present invention if it contains a tertiary amine or quaternary ammonium in the repeating unit. However, it should be noted that it may exhibit cytotoxicity or cytotoxicity. In the present invention, it is preferable to use hexadimethrine bromide (HDB) having a repeating structure of quaternary ammonium. Furthermore, polyethyleneimine (PEI) may be added. When used together with HDB, PEI acts as an auxiliary in transporting proteins into cells. Alternatively, it is also preferable to use protamine as a cationic carrier molecule. The molecular weight range of the cationic polymer is preferably 1,000 to 1,000,000, more preferably 1,000 to 100,000, in terms of weight average molecular weight. PEI may be linear PEI, branched PEI, or may contain a crosslinked structure. For example, linear PEI obtained by deacylation of linear poly(2-acyl oxazoline) synthesized by ring-opening polymerization of 2-alkyl-2-oxazoline can be used.

[0028] The anionic carrier molecule that can be preferably used in the complex of the present invention is most preferably a boron cluster (closo-borate) salt represented by the following general formula 1. In the following general formula 1, M represents a cation selected from lithium, sodium, potassium, and cesium. B represents a boron atom, X represents hydrogen or a halogen atom (F to I), and n represents 10 or 12.

[0029] (chemical 1) M 2+ (B n X n ) 2- (General formula 1)

[0030] The boron cluster salt represented by the general formula 1 can be produced by utilizing a known method for synthesis (for example, Non-Patent Document 6 "I. Tiririris et al., "The Crystal Structure of Dicaesium-Dodekahalogeno-closo-Dodekaborate Cs2[B12X12] (X = Cl, Br, I) and its Hydrate", Zeitschrift fur anorganische und allgemeine Chemie, 630(11):1555-1563 (2004)."). In this case, the number n of boron atoms forming the boron cluster portion is preferably 12 because it allows stable production with good yield, but it may also be that a component having a different number of atoms is contained. In addition, as X in the above formula, hydrogen and halogens ranging from fluorine to iodine are bonded to boron atoms to form boron cluster portions. Unlike when going from fluorine to iodine, the hydrophobicity of the boron cluster increases and the electron density of the negative charge of the anion decreases, so that in addition to electrostatic interactions, hydrophobic interactions with lipid membranes and proteins become stronger. In the present invention, however, the halogen bromine is most preferably used since it does not show any cytotoxicity or cytotoxicity and has excellent protein transport properties.

[0031] In the present invention, it has been found that by using the above boron cluster salt in combination with a cationic polymer, it is possible to deliver various proteins into cells and express their activity in the cytoplasm. Conventionally, various cationic polymers have been used as gene transfection vectors. In the present invention, polyethyleneimine (PEI), which has conventionally been used for gene transfection, can be particularly preferably used. PEI is known to easily bind to the negatively charged cell membrane surface and to have only slight cytotoxicity. It has been known that after a complex of PEI and DNA is taken up into a cell by endocytosis, the secondary amine of the main chain of polyethyleneimine is protonated on the weak acidic side in the endosome, causing swelling, and destroying the endosomal membrane by the so-called proton sponge action, thereby releasing the encapsulated DNA into the cytoplasm. The present invention is also characterized in that cationic carrier molecules such as PEI are transported together with proteins in the cytosol in the cell in a form encapsulated in endosomes. In order to enable the escape of proteins from endosomes, the presence of cationic carrier molecules such as PEI is essential in the present invention.

[0032] In the present invention, it may be necessary for the complex consisting of cationic carrier molecules, anionic carrier molecules, and proteins to have the same pharmacokinetics of the components in the body until they are taken up by target cells in the body when the complex is applied to actual clinical applications. As mentioned above, the components constituting the complex are reversibly bound to each other by electrostatic interactions, hydrophobic interactions, etc., but the environments in which the complex is placed are significantly different between when it is in interstitial fluid and when it is in the cytosol, and this is thought to cause a significant change in the stability of the complex. In the interstitial fluid, plasma proteins such as albumin are dissolved at a concentration of 10% or less together with various inorganic ions, while in the cytosol, a wide variety of proteins and low molecular weight compounds are contained at high concentrations surrounded by various intracellular structures. In the interstitial fluid, the stability of the complex is similar to that of polymer colloids in dilute solutions, and is easily prevented from aggregation and decomposition due to surface charges and excluded volume effects, but in the cytosol, the interactions that hold the complexes together are lost due to interactions with surrounding proteins, etc., and it is expected that they will dissociate. Therefore, the complex of the present invention is characterized in that, since the protein remains integrated as a complex until it is transported to the target cell, the pharmacokinetics of the components can be consistent, but the integration does not necessarily need to be maintained after introduction into the cytosol, and the protein can be released from the complex and exert its original structure and function within the cytosol.

[0033] As described above, in the present invention, a complex consisting of a cationic carrier molecule such as PEI bound to a transport protein and an anionic carrier molecule is characterized in that it is transported in the form of being encapsulated in an endosome into the cytosol in a cell by endocytosis. When the complex encapsulated and taken up in an endosome in a cell generates defects in the endosomal membrane due to swelling caused by neutralization of PEI, it is expected that the components of the complex will gradually diffuse into the cytosol. In the cytosol, PEI is surrounded by high concentrations of proteins, amino acids, and other hydrophilic components, and it is thought that the binding with the transported protein will gradually be lost. As shown in the examples described later, when a fluorescently labeled nuclear transport protein was actually used as a transport protein, the presence of the fluorescently labeled protein was confirmed in the cell nucleus, confirming that the transport protein actually escaped the endosome, diffused into the cytosol, and then transported into the nucleus.

[0034] In the present invention, the protein that can be used in the complex is preferably an antibody. It is expected that the complex of the present invention will be specifically taken up by specific target cells such as tumor cells, and will bind to antibody receptors in the tumor cells, activating the immune signaling system and producing inflammatory cytokines. This system is expected to be used as an antibody drug that specifically recognizes tumor cells and induces their cell death. Furthermore, the complex can also be used as an antibody-drug conjugate (ADC) that simultaneously contains various anticancer drugs together with the antibody.

[0035] Alternatively, as another example, for use in genome editing, a method can be preferably used in which a guide RNA (gRNA) used in CRISPR (Clustered regularly interspaced short palindromic repeats) / Cas9 (CRISPR-associated proteins) is mixed with a Cas9 protein (DNA cleavage enzyme) to be transported into a cell to form a nucleic acid-protein complex (RNP), which is then incorporated into the complex of the present invention for use. Since the Cas9 protein is a relatively large protein with a molecular weight of more than 100 kD, it has been difficult to transport it into a cell using conventional techniques, but by combining the cationic carrier molecule of the present invention with the anionic carrier molecule to form a complex, it can be incorporated into a target cell by an endocytosis mechanism. By knocking out a gene in a specific target cell, it is expected to be used in cancer immunotherapy and gene therapy for various intractable diseases.

[0036] In the present invention, the preparation of the complex and the method of transporting it into the cells are preferably carried out as follows. First, a first step is provided in which a solution in which a cationic carrier molecule is dissolved, a solution in which an anionic carrier molecule is dissolved, and a solution in which a protein is dissolved are prepared, and these are added to each other and mixed to prepare an intracellular protein delivery agent. In this case, in the case of in vivo cell experiments, each solution is preferably dissolved in a simple DMEM medium that does not contain serum, and in the case of injection into a living body, each solution is preferably dissolved in physiological saline. Next, as a second step of contacting the prepared intracellular protein delivery agent with the cell surface, in in vitro experiments, the delivery agent is added to the medium in a container in which cells are cultured and diffused on the cell surface, and when introducing the intracellular protein delivery agent into the living body, a method such as locally injecting the delivery agent into the site where the target cells are present via a syringe or the like can be adopted.

[0037] Next, a third step is provided in which the intracellular protein delivery agent is introduced into the cytoplasm in a state encapsulated in an endosomal membrane by endocytosis, with a rest period ranging from several minutes to several hours after the introduction of the delivery agent. When endocytosis is clathrin-dependent, openings are expressed on about 2% of the cell surface and their life span is about 1 minute, so that endocytosis can be effective in all cells, the concentration of the delivery agent and the rest time in this step are important factors. Furthermore, in this third step, the cells may be in a normal nutrient medium, or the delivery agent may be directly injected into the interstitial fluid of the living body.

[0038] As the next step, a fourth step is provided in which the intracellular protein delivery agent escapes the endosomal membrane and diffuses into the cytoplasm, but since this step is performed in parallel with the cell culture, if the medium is replaced with a fresh one or injected into a living body, a sealing treatment of the opening is performed. Furthermore, when the purpose is transport from the cytosol into the cell nucleus, a fifth step is provided in which an additional time is provided to ensure the time required for the diffusion of the transport protein contained in the delivery agent.

[0039] In the present invention, there are preferred ranges for the respective ratios of cationic carrier molecules, anionic carrier molecules, and proteins used in the complex. When the protein to be transported is taken as 100 parts by mass, the cationic carrier molecules are preferably used in the range of 5 to 500 parts by mass, and more preferably in the range of 5 to 50 parts by mass. Similarly, the anionic carrier molecules are preferably used in the range of 10 to 500 parts by mass relative to 100 parts by mass of the transport protein.

[0040] An example of an embodiment of the present invention will be described in detail below with reference to the drawings, but the present invention is not limited to these examples and illustrated examples, and many modifications and variations are possible. Note that percentages in the examples are based on mass unless otherwise specified. EXAMPLES

[0041] We investigated the delivery of nuclear localizing green fluorescent protein (NLS-EGFP) to the cell nucleus. A gene sequence encoding NLS-EGFP, which binds NLS (nuclear localization signal: in this example, c-Myc-derived NLS (amino acid sequence PAAKRVKLD)) to the N-terminus of EGFP (Enhanced Green Fluorescent Protein; improved green fluorescent protein with excitation wavelength of 488 nm and emission wavelength of 507 nm), was inserted into a plasmid (Addgene plasmid vector, backbone: pET15b vector), and a sample was expressed and purified using E. coli. A solution was prepared by dissolving 8 μg of NLS-EGFP (transport protein) in 250 μL of DMEM(-) (Dulbecco's Modified Eagle Medium: serum-free). 12 Br 12 According to the method described in Non-Patent Document 6, cesium dodecaborate (CsB 12 H 12 67.8μg of polyethylenimine (PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000) 24765-1, Polyscience) was dissolved in DMEM(-) in separate containers, and then the two were mixed and dissolved in 250μL of DMEM(-). The former protein solution and the latter mixed solution were mixed, shaken, and left at room temperature for 5 minutes. The complex prepared as an intracellular protein delivery agent in this example was prepared as a 500μL DMEM(-) solution containing PEI (1.5μg, 75nM), boron cluster salt (67.8μg, 100μM), and NLS-EGFP (8μg, 0.6μM) (flow S01 in Figure 3).

[0042] 1 × 10 U-2 OS cells (92022711; European Collection of Authenticated Cell Cultures, Public Health England, London, UK) 5 The cells were introduced into a 24-well plate at a cell count of 100 μL / well. A cover glass was placed in each well of the plate, and the cells were cultured on the glass surface. The cells were washed twice with PBS(-), and then 500 μL of DMEM(-) containing the complex prepared above was added to each well (Flow S02 in Figure 3). After culturing for 1 hour in a CO2 incubator adjusted to 37°C, the medium was removed and the wells were washed twice with PBS(-) (Flow S03 in Figure 3). Next, DMEM(+) medium containing 10% FBS was added to each well, and the wells were cultured at 37°C for 2 hours in a CO2 incubator (Flows S04 and S05 in Figure 3). After washing twice with PBS(-), the cells were fixed with 4% paraformaldehyde. The cells were washed twice with PBS(-) and then stained with a solution of DAPI dissolved in PBS containing Triton X-100 as a nuclear staining solution, washed twice with PBS(-), and embedded in ProLong Glass.

[0043] The obtained samples were observed using a laser scanning confocal fluorescence microscope (model name: LSM700, manufacturer name: ZEISS). The observed images are shown in FIG. 2. FIG. 2 shows a fluorescence microscope photograph showing that green fluorescent protein was delivered into the cell nucleus. This result demonstrated that by using the intracellular protein delivery agent of the present invention, not only can the target protein be delivered into the cell, but also the protein escapes from the endosome and effectively migrates into the nucleus. EXAMPLES

[0044] A rhodamine-labeled secondary antibody (Goat Anti-Rat IgG Antibody, Rhodamine conjugate, AP136R, Sigma-Aldrich) was prepared by immunizing a goat to detect rat IgG, and was used to deliver the antibody into the cytosol of U-2 OS cells in the same manner as in Example 1. A solution was prepared by dissolving 4 μg of the secondary antibody in 250 μL of DMEM(-) (Dulbecco's Modified Eagle Medium: serum-free). Cs2B 12 Br 12 67.8 μg of PEI and 1.5 μg of polyethyleneimine (PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000) 24765-1, Polyscience) were dissolved in 250 μL of DMEM(-). Both solutions were mixed and shaken, and the mixture was allowed to stand at room temperature for 5 minutes. The complex prepared as an intracellular delivery agent in this example was prepared as a 500 μL DMEM(-) solution containing PEI (1.5 μg, 75 nM), boron cluster salt (67.8 μg, 100 μM), and the above secondary antibody (4 μg).

[0045] 1 × 10 U-2 OS cells (92022711; European Collection of Authenticated Cell Cultures, Public Health England, London, UK) 5The cells were introduced into a 24-well plate at a cell number of 100 μL. A cover glass was placed in each well of the plate, and cell culture was performed on the glass surface. The cells were washed twice with PBS(-), and 500 μL of DMEM(-) containing the complex prepared above was added to each well. After culturing for 1 hour in a CO2 incubator adjusted to 37°C, the medium was removed and washed twice with PBS(-). Next, DMEM(+) medium containing 10% FBS was added to each well and cultured at 37°C for 2 hours in a CO2 incubator. After washing twice with PBS(-), the cells were fixed with 4% paraformaldehyde. The cells were washed twice with PBS(-), stained with a solution of DAPI dissolved in PBS containing Triton X-100 as a nuclear staining solution, washed twice with PBS(-), and embedded in ProLong Glass.

[0046] The obtained sample was observed using a laser scanning confocal fluorescence microscope (model name: LSM700, manufacturer name: ZEISS). The observed image is shown in FIG. 4. FIG. 4 shows a fluorescence microscope photograph showing that the secondary antibody was delivered into the cell. This result demonstrated that the antibody was delivered into the cell by using the intracellular protein delivery agent of the present invention. From FIG. 4, it was confirmed that the antibody was not distributed throughout the cytosol, but accumulated at a high concentration on one side of the cell. It is considered that the antibody was encapsulated in the endosome by endocytosis and transported into the cytosol, but from this result it cannot be determined whether the antibody escaped from the endosome. Since antibodies easily aggregate due to changes in the pH, salt concentration, temperature, etc. of the solution containing them, it is possible that the antibody molecules aggregated with each other at the stage of forming a complex using the delivery agent of the present invention, and were introduced into the cytosol in an aggregated form. In that case, it is possible that the antibody molecules were observed to be ubiquitously present on one side of the cell due to the prevention of diffusion in the cytosol. With regard to the introduction of the antibody into the cytosol, it is considered important to optimize various conditions such as the concentration, temperature, and pH when preparing the complex of the present invention. EXAMPLES

[0047] We delivered the nuclear-localized green fluorescent protein (EGFP-NLS) to the cell nucleus. 12 Br 12 100 μM, 32 μM (6 μg) of hexadimethrine bromide (HDB, manufactured by Merck) as a quaternary ammonium as a cationic polymer, and 6 μg of polyethyleneimine (PEI, manufactured by Polyscience) as a secondary amine as a polymer were added to 500 μL of DMEM(-) and vortexed. 8 μg of EGFP-NLS was added thereto, vortexed, and allowed to stand at room temperature for 15 minutes. The complex prepared as an intracellular protein delivery agent in this example was HDB (6 μg, 32 μM), PEI (6 μg, 0.3 μM), boron cluster salt Cs2B 12 Br 12 (67.8 μg, 100 μM) and EGFP-NLS (8 μg, 0.6 μM) in 500 μL of DMEM(-).

[0048] 1 × 10 U-2 OS cells 5 The cells were introduced into a 24-well plate at a cell number of 100 μL. At this time, a cover glass was placed in each well of the plate, and cell culture was performed on the glass surface. The cells were washed twice with PBS(-), and 500 μL of DMEM(-) containing the complex prepared above was added to each well. After culturing for 1 hour in a CO2 incubator adjusted to 37°C, the medium was removed and washed twice with PBS(-). Next, DMEM(+) medium containing 10% FBS was added to each well and cultured at 37°C for 3 hours in a CO2 incubator. After washing twice with PBS(-), the cells were fixed with 4% paraformaldehyde. After washing twice with PBS(-), the cells were stained with a solution of DAPI dissolved in PBS containing Triton X-100 as a nuclear staining solution, washed twice with PBS(-), and embedded in ProLong Glass. The obtained sample was observed using a laser scanning confocal fluorescence microscope in the same manner as in Example 1. The observed image is shown in FIG. 5(A). A graph showing the delivery efficiency is shown in FIG. 5(B). FIG. 5(A) shows a fluorescence microscope photograph showing that green fluorescent protein is delivered into the cell nucleus. The results in FIG. 5 show that the protein is delivered into the cell more efficiently by adding HDB and PEI as boron clusters and cationic polymers to the protein. In other words, it was demonstrated that the intracellular protein delivery agent of the present invention can not only deliver the target protein into the cell, but also allows the protein to escape from the endosome and migrate effectively into the nucleus. EXAMPLES

[0049] Lysozyme (19499-04, Nacalai Tesque) labeled with FITC (FITC-Lysozyme) and the rhodamine-labeled secondary antibody used in Example 2 were delivered to the cytosol. 12 Br 12 Or Cs2B 12 Br 12 100μM, 50μM (9.4μg) or 32μM (6μg) of hexadimethrine bromide (HDB, Merck) as a quaternary ammonium as a cationic polymer, and 1.5μg of polyethyleneimine (PEI, Polyscience) as a secondary amine as a polymer were added to 500μL of DMEM(-) and vortexed. 7.2μg of FITC-Lysozyme or 4μg of secondary antibody were added thereto, vortexed, and allowed to stand at room temperature for 15 minutes. The complex prepared as an intracellular protein delivery agent in this example was HDB (9.4μg, 50μM), PEI (1.5μg, 75nM), boron cluster salt Na2B 12 Br 12 (56.7 μg, 100 μM) and FITC-Lysozyme (7.2 μg, 1 μM) in 500 μL of DMEM(-), or HDB (6 μg, 32 μM), PEI (1.5 μg, 75 nM), and boron cluster salt Cs2B 12 Br 12(67.8 μg, 100 μM) and secondary antibody (4 μg) in 500 μL of DMEM(-).

[0050] 1 × 10 U-2 OS cells 5 The cells were introduced into a 24-well plate at a cell number of 100 μL. At this time, a cover glass was placed in each well of the plate, and cell culture was performed on the glass surface. The cells were washed twice with PBS(-), and then 500 μL of DMEM(-) containing the complex prepared above was added to each well. After culturing for 1 hour in a CO2 incubator adjusted to 37°C, the medium was removed and washed twice with PBS(-). Next, DMEM(+) medium containing 10% FBS was added to each well and cultured at 37°C for 24 hours in a CO2 incubator. After washing twice with PBS(-), the cells were fixed with 4% paraformaldehyde. After washing twice with PBS(-), the cells were stained with a solution of DAPI dissolved in PBS containing Triton X-100 as a nuclear staining solution, washed twice with PBS(-), and embedded in a glycerol-based mounting agent. The obtained samples were observed using a laser scanning confocal fluorescence microscope in the same manner as in Example 1. The observed images are shown in Figs. 6 and 7. Figs. 6 and 7 show fluorescence microscopy photographs showing that each protein was delivered to the cytosol. From the results in Figs. 6 and 7, the protein was delivered into the cell more efficiently by adding HDB and PEI as boron clusters and cationic polymers to the protein. In other words, it was demonstrated that the intracellular protein delivery agent of the present invention not only allowed the target protein to be delivered into the cell, but also allowed the protein to escape from the endosome and effectively migrate to the cytosol. EXAMPLES

[0051] FITC-labeled bovine serum albumin (FITC-BSA, A9771, Sigma) was delivered to the cytosol. 12 Br 12100 μM and 12 μg of protamine (293-18, Nacalai Tesque) as a cationic molecule were added to 500 μL of DMEM(-) and vortexed. 16.5 μg of FITC-BSA was added thereto, vortexed, and allowed to stand at room temperature for 15 minutes. The complex prepared as an intracellular protein delivery agent in this example was protamine (12 μg), boron cluster salt Cs2B 12 Br 12 (67.8 μg, 100 μM) and FITC-BSA (16.5 μg, 0.5 μM) in 500 μL of DMEM(-).

[0052] 1 × 10 U-2 OS cells 5 The cells were introduced into a 24-well plate at a cell number of 100 μL. At this time, a cover glass was placed in each well of the plate, and cell culture was performed on the glass surface. The cells were washed twice with PBS(-), and 500 μL of DMEM(-) containing the complex prepared above was added to each well. After culturing for 1 hour in a CO2 incubator adjusted to 37°C, the medium was removed and washed twice with PBS(-). Next, DMEM(+) medium containing 10% FBS was added to each well and cultured at 37°C for 3 hours in a CO2 incubator. After washing twice with PBS(-), the cells were fixed with 4% paraformaldehyde. The cells were washed twice with PBS(-), stained with a solution of Hoechst33342 dissolved in PBS as a nuclear staining solution, washed twice with PBS(-), and embedded in a glycerol-based mounting agent. The obtained sample was observed using a laser scanning confocal fluorescence microscope in the same manner as in Example 1. The observed image is shown in FIG. 8. FIG. 8 shows a fluorescence microscope photograph showing that FITC-BSA was delivered to the cytosol. The results in FIG. 8 show that the protein was delivered into the cell more efficiently by adding a boron cluster and protamine as a cationic molecule to the protein. In other words, it was demonstrated that the use of the intracellular protein delivery agent of the present invention not only allowed the target protein to be delivered into the cell, but also allowed the protein to escape from the endosome and effectively migrate to the cytosol. EXAMPLES

[0053] The boron cluster salt Cs2B was used as the anionic carrier molecule at the same concentration as in the above examples. 12 Br 12 An aqueous solution containing HDB as a cationic carrier molecule and the resulting solution was irradiated with ultrasonic waves for 20 seconds. To this solution, bovine serum albumin (BSA) was added as a protein at the same concentration as in the above-mentioned Example, and the solution was irradiated with ultrasonic waves for another 20 seconds. The average particle size of the resulting aqueous solution was measured based on the scattering intensity distribution. The average particle size was measured using dynamic light scattering (DLS), and the graph of the measurement results is shown in FIG. 9. From the results in FIG. 9, the formation of complexes with an average particle size of 100 nm was confirmed, and it was found that these complexes function as protein delivery agents. [Industrial Applicability]

[0054] Since the present invention can efficiently deliver various proteins into cells, it can be suitably used for antibody drugs and CRISPR / Cas9 for genome editing.

Claims

1. An intracellular protein delivery agent which is a complex consisting of at least a cationic carrier molecule and an anionic carrier molecule, which are bonded to each other by at least ionic bonds and hydrophobic interactions.

2. 2. The intracellular protein delivery agent according to claim 1, wherein the cationic carrier molecule, the anionic carrier molecule, and the protein are in the form of a complex that is bonded to each other by at least ionic bonds and hydrophobic interactions.

3. The intracellular protein delivery agent according to claim 1 or 2, wherein the cationic carrier molecule is an oligomer or polymer having a cationic group as a repeating unit.

4. The intracellular protein delivery agent according to claim 1 or 2, wherein the cationic carrier molecule is protamine.

5. The intracellular protein delivery agent according to claim 1 or 2, wherein the cationic carrier molecule is hexadimethrine bromide.

6. The intracellular protein delivery agent according to claim 5 , further comprising polyethyleneimine as the cationic carrier molecule.

7. The intracellular protein delivery agent according to claim 2 , wherein the protein is an antibody, an enzyme, or a nucleic acid-protein complex for CRISPR / Cas9.

8. The intracellular protein delivery agent according to any one of claims 1, 2 and 7, wherein the anionic carrier molecule is a boron cluster salt represented by the following general formula 1: (Chem.1) M ２＋ (B ｎ X ｎ ) ２－ (General formula 1) (In the above formula, M represents a cation selected from lithium, sodium, potassium, and cesium, B represents a boron atom, X represents hydrogen or a halogen atom (F to I), and n represents 10 or 12).

9. A first step of preparing an intracellular protein delivery agent by mixing a solution in which a cationic carrier molecule is dissolved, a solution in which an anionic carrier molecule is dissolved, and a solution in which a protein is dissolved; a second step of contacting the intracellular protein delivery agent with a cell surface; a third step in which the intracellular protein delivery agent is introduced into the cytoplasm by endocytosis in a state encapsulated in an endosomal membrane; a fourth step in which the intracellular protein delivery agent escapes the endosomal membrane and diffuses into the cytoplasm; The present invention relates to a method for introducing a protein into an cell.

10. The method for intracellular protein introduction described in claim 9, further comprising a fifth step in which the intracellular protein delivery agent diffuses into a cell nucleus.

Images