TECHNICAL FIELD

The present invention relates to a method for introducing an exogenous substance into a cell and a material used in that method. More particularly, the present invention relates to a method for introducing an exogenous substance into a cell using uptake of an exosome by macropinocytosis, and a material used in that method.

Furthermore, the present application claims priority on the basis of Japanese Patent Application No. 2014-230472 filed on Nov. 13, 2014, the contents of which are incorporated herein in their entirety by reference.

BACKGROUND ART

The development of technology for spatially, chronologically and quantitatively controlling the distribution of a drug in the body is an extremely important issue in the field of pharmaceuticals. Preferably controlling the distribution of a drug in the body makes it possible to, for example, enhance drug efficacy, mitigate adverse side effects, decrease the required number of administrations and reduce costs. Technology for spatially, chronologically and quantitatively controlling the distribution of a drug in the body in this manner is generally referred to as a drug delivery system (DDS), and extensive research is currently being conducted in this field.

Attention has recently been focused on technology relating to DDS at the cellular level, and typically technology for efficiently introducing an exogenous substance into a target cell. The application of this technology makes it possible to efficiently supply (or allow to act) a target drug on the causative cell of a disease, for example. In particular, if it were possible to only allow a drug having potent adverse side effects on normal cells (such as an anticancer drug) to efficiently act on target cells (such as cancer cells), a high level of efficacy would be able to be realized while reducing adverse side effects.

In addition, this technology relating to DDS at the cellular level is also attracting considerable attention in the field of regenerative medicine. For example, the application of this technology enables a reprogramming factor, differentiation-inducing factor, etc., to be efficiently introduced into a target cell. In addition, when grafting a graft material for regenerative medicine (such as stem cells used in regenerative medicine or cells and tissue induced to differentiate from these stem cells) into the body, this technology can be applied to efficiently supply factors to the graft material that are required until the graft material becomes established in the body.

Patent Literature 1 to 3 describe technologies relating to DDS at the cellular level in the form of techniques for introducing a desired substance (and typically, a drug) into a cell using various carrier substances.

CITATION LIST

Patent Literature

Patent Literature 1: Japanese Patent Application Laid-open No. 2013-121986Patent Literature 2: Japanese Patent Application Laid-open No. 2013-063926Patent Literature 3: Japanese Translation of PCT Application No. 2003-517886

Non Patent Literature

Non Patent Literature 1: Chemistry & Biology, Vol. 19, 2012, pp. 1437-1446Non Patent Literature 2: The Journal of Biological Chemistry, Vol. 289, 2014, pp. 22258-22267Non Patent Literature 3: Journal of Cell Science, Vol. 94, 1989, pp. 135-142

SUMMARY OF INVENTION

However, in the conventional DDS described in Patent Literature 1 to 3, for example, compounds used in vitro are typically used as carrier substances for introducing an exogenous substance into a cell. When such an in vitro compound is used, there are cases in which adequate effects are unable to be demonstrated due to cytotoxicity attributable to the carrier or an immune response to the carrier.

Consequently, attempts have been made to establish a technology for introducing an exogenous substance into a cell using a bio-derived substance.

With the foregoing in view, the present invention was conceived for the purpose of providing a novel material used to introduce an exogenous substance into a cell. More specifically, an object of the present invention is to establish a technology for introducing an exogenous substance into the body using a bio-derived substance. In addition, another object of the present invention is to provide a method for introducing an exogenous substance into a target cell using that material.

The inventors of the present invention focused on exosomes, which are widely distributed throughout the body, as a means for achieving these objects. Exosomes refer to vesicles present in the body (and typically, in body fluids) that are formed from a lipid bilayer membrane. As a result of conducting extensive research on the mechanism by which exosomes are taken up into cells, the inventors of the present invention found that exogenous substances contained in exosomes can be preferably introduced into a target cell by inducing macropinocytosis in a target cell. Moreover, the inventors of the present invention newly found that the uptake of an exosome into a target cell is promoted by supplying the target cell with an exosome containing a substance that induces macropinocytosis. The inventors of the present invention completed the present invention on the basis of these findings.

Non Patent Literature 1 describes a phenomenon in which macropinocytosis is activated by supplying a cell with a substance that stimulates a specific receptor, while Non Patent Literature 2 describes a phenomenon in which exosomes are taken up into cells by macropinocytosis. However, there are no disclosures or suggestions that an exogenous substance contained in an exosome can be preferably introduced into a target cell by inducing macropinocytosis or that the uptake of an exosome into a cell is promoted by supplying the cell with an exosome containing a substance that induces macropinocytosis.

In order to realize these objects, the present invention provides an exosome having the ability to introduce an exogenous substance into a target cell (and this exosome is also referred to as an “exosome for introducing an exogenous substance”) when the exosome is supplied to the target cell (and typically, when added to a medium used to culture the target cell).

Namely, the exosome disclosed herein (exosome for introducing an exogenous substance) is an exosome used to introduce an exogenous substance into a target cell from outside the target cell, containing:

one type or two or more types of an exogenous substance; and

a substance that induces macropinocytosis in the target cell.

The exosome for introducing an exogenous substance disclosed herein is characterized by containing a substance that induces macropinocytosis in the exosome along with an exogenous substance. As a result thereof, macropinocytosis can be induced (activated) in a target cell and uptake of the exosome by the target cell can be promoted. Thus, an exogenous substance contained in an exosome for introducing an exogenous substance can be efficiently introduced into a target cell.

Here, an exosome refers to a bio-derived substance. Consequently, cytotoxicity attributable to the exosome and an immune response to the exosome can be reduced in comparison with a substance derived from an in vitro compound. Consequently, the exosome is preferable as a carrier for introducing an exogenous substance.

In addition, since the exosome for introducing an exogenous substance disclosed herein contains a target exogenous substance and a substance that induces macropinocytosis therein, it is not necessary to provide or manipulate the substance inducing macropinocytosis and the exosome containing the exogenous substance, or carry out procedures for mixing these substances. Consequently, a target exogenous substance can be introduced into a target cell by a simple procedure including supplying an exosome for introducing an exogenous substance into the target cell.

In addition, exosomes are vesicles that are able to stably maintain the structure thereof in cell culture broth. In addition, they are resistant to decomposition and are stable in the body (such as in blood). Consequently, by containing a substance that induces macropinocytosis and an exogenous substance in an exosome, the decomposition (including metabolism and deactivation) thereof can be reduced. For example, metabolism of a drug in the liver, excretion as waste and elimination by immune cells can be preferably reduced (avoided). Thus, the exosome for introducing an exogenous substance disclosed herein is able to stably supply a substance inducing macropinocytosis and an exogenous substance into a cell (and particularly a cell in the body).

Consequently, the exosome for introducing an exogenous substance disclosed herein is preferable as a component of a composition used in an application for introducing an exogenous substance into a target cell. In addition, it can also be used in a method for introducing an exogenous substance into a target cell.

In a preferable mode of the exosome (exosome for introducing an exogenous substance) disclosed herein, the substance that induces macropinocytosis in the target cell is a substance that stimulates (activates) epidermal growth factor receptor (EGFR) or a substance that stimulates (activates) CXC chemokine receptor 4 (also referred to as CXCR4, CD184 or fusin).

Epidermal growth factor receptor and CXC chemokine receptor 4 are typical examples of receptors capable of inducing macropinocytosis following stimulation thereof. Consequently, substances that stimulate (activate) epidermal growth factor receptor and substances that stimulate (activate) CXC chemokine receptor 4 are preferable as substances for inducing macropinocytosis.

In particular, an exosome containing EGF or an analog thereof or containing stromal cell-derived factor (SDF) or an analog thereof for the substance that induces macropinocytosis in a target cell is a preferable mode of the exosome (exosome for introducing an exogenous substance) disclosed herein. EGF or an analog thereof is a substance that preferably stimulates epidermal growth factor receptor, while SDF or an analog thereof is a substance that preferably stimulates CXC chemokine receptor 4.

In general, chemokines have four cysteine residues (C) preserved within a molecule thereof, and are classified into four subfamilies (C, CC, CXC and CX3C) according to differences in the relationship between the two cysteine residues on the N-terminal side and other amino acid residues. Among these, CXC chemokines refer to chemokines belonging to the subfamily having an amino acid sequence (“C-X-C” sequence) in which a single arbitrary amino acid residue (X) is present between the two cysteine residues (C) on the N-terminal side.

In a preferable mode of the exosome (exosome for introducing an exogenous substance) disclosed herein, the exogenous substance has pharmacological activity. As a result of employing this configuration, a substance having pharmacological activity can be efficiently supplied to a target cell. As a result, the pharmacological effect of the substance having pharmacological activity can be demonstrated at a high level.

In a preferable mode of the exosome (exosome for introducing an exogenous substance) disclosed herein, the target cell is a human tumor cell and the exogenous substance is a compound having antitumor activity. Many compounds having antitumor activity typically have potent adverse side effects on normal cells. According to this mode, since a compound having antitumor activity can be efficiently supplied to a human tumor cell, antitumor activity can be demonstrated at a high level on the tumor cell while reducing adverse side effects on normal cells attributable to the compound having antitumor activity.

In addition, in another aspect thereof, the present invention provides a composition (pharmaceutical composition) used to introduce an exogenous substance into a target cell from outside the target cell (this composition may also be referred to as a “composition for introducing an exogenous substance”), comprising:

one type or two or more types of a pharmaceutically acceptable carrier;

a substance that induces macropinocytosis in the target cell; and

an exosome containing one type or two or more types of an exogenous substance.

Since the composition having this configuration (composition for introducing an exogenous substance) contains a substance that induces macropinocytosis, uptake of an exosome into a target cell can be preferably promoted. As a result, an exogenous substance contained in the exosome can be efficiently introduced into a target cell.

In addition, in another aspect thereof, the present invention provides a composition (pharmaceutical composition) used to introduce an exogenous substance into a target cell from outside the target cell (composition for introducing an exogenous substance), comprising:

one type or two or more types of a pharmaceutically acceptable carrier; and

an endosome containing a substance that induces macropinocytosis in the target cell and one type or two or more types of an exogenous substance.

Since the composition of this configuration (composition for introducing an exogenous substance) contains a substance that induces macropinocytosis in an exosome, uptake of the exosome into the target cell can be preferably promoted. As a result, the exogenous substance contained in the exosome can be efficiently introduced into the target cell. Moreover, since a substance that induces macropinocytosis in the target cell is contained within the exosome, the substance that induces macropinocytosis can be stably supplied to the target cell.

In addition, in another aspect thereof, the present invention provides a method for introducing an exogenous substance into a target cell in vitro, comprising:

preparing a cell culture containing the target cell; and

supplying an exosome containing a substance that induces macropinocytosis in the target cell and one type or two or more types of an exogenous substance to the cell culture.

According to this method for introducing an exogenous substance, an exogenous substance can be efficiently introduced into a target cell by an easy method including supplying an exosome to a culture containing the target cell (and typically, the medium used to culture the target cell). In addition, by using an exosome that is a bio-derived substance as a carrier for introducing an exogenous substance into a cell, cytotoxicity due to the carrier can be reduced.

