JP2006158314A - Carrier agent for introducing compound into cell and method for introducing compound into cell therewith - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for introducing a compound into a cell, by which the compound having a relatively low mol. wt. including a low mol. wt. nucleic acid can simply, safely and efficiently be introduced into the cell in low toxicity without needing special equipment, and to provide a compound carrier therefor. <P>SOLUTION: The carrier agent for introducing the compound in the cell comprises a compound represented by formula (I) [(m) is 1 or 2; (n) is an integer of 2 to 6]. A complex of the carrier with a compound to be introduced (preferably a low molecular nucleic acid such as siRNA). A method for introducing the compound into the cell comprises bringing the complex into contact with the cell. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a carrier agent comprising a cationic lipid, which is suitable for efficiently introducing a relatively low molecular weight compound such as a low-molecular nucleic acid such as an antisense oligonucleotide into a cell, and the carrier agent. The present invention relates to a method of introducing the target compound into cells.

In recent years, various diseases such as cancer, cranial nerve disease, AIDS, and genetic diseases have been energetically clarified, and many causative genes and related genes have been clarified. Along with this, gene therapy methods targeting genes involved in diseases are attracting great expectations. Gene therapy methods can be broadly classified into those that are intended to supplement missing genetic information and those that are intended to suppress the expression of disease-causing genes such as genes essential for the growth of cancer cells. However, in the latter, a low molecular nucleic acid complementary to the mRNA of the target gene (eg, oligo DNA / RNA, modified oligonucleotide, peptide nucleic acid (PNA), etc.) is administered to specifically suppress the expression of the target gene. Antisense methods have been devised. In particular, RNA interference (RNAi) technology, represented by short interfering RNA (siRNA), was selected as the first place in the 2002 Breakthrough of the Year (Science magazine). The speed of drug development is greatly accelerated.
Since RNAi-based antisense technology is now being used extensively for basic research and new drug development, it can be applied to cultured cells (in vitro, ex vivo) and in vivo tissues (in vivo). Development of a technique for increasing the efficiency of molecular nucleic acid introduction, that is, a carrier agent capable of achieving higher frequency intracellular introduction is desired.

As a conventional method for introducing a low molecular nucleic acid into a cell, a method using a calcium phosphate method, a DEAE-dextran reagent, etc., Lipofectin (trade name), Lipofectamine (trade name), Lipofectamine 2000 (trade name), etc. However, in many cases, the introduction efficiency is low and cytotoxicity is observed. On the other hand, the microinjection method, electroporation method, gene gun (particle gun) method, etc. have relatively high introduction efficiency, but problems such as the need to purchase expensive equipment and skill in operation. Therefore, it is not suitable for processing a large amount of cells. Furthermore, methods using viruses such as adenovirus have problems such as operability such as preparation of virus-sensitive cells and virus infection to researchers.
The present inventors have already made a bilayer membrane of an amphipathic molecule at a phase transition temperature close to a biological membrane temperature as a method that is easy to operate, has no cytotoxicity, and can efficiently introduce a gene into a cell. Although the method using the synthetic bilayer membrane obtained by this method has been developed, this method mainly uses about 2 kb or more of high-molecular nucleic acid (for example, in the case of gene transfer for complementing a defective gene) in a cell efficiently. Intracellular introduction of low-molecular nucleic acids such as siRNA and antisense oligo DNA, or other compounds having a molecular weight equal to or lower than that such as proteins, bioactive peptides and chemotherapeutic agents For, there was a need for further efficiency improvements.
Japanese Patent Publication No. 7-20429

  Therefore, an object of the present invention is to introduce a relatively low molecular weight compound such as a low-molecular-weight nucleic acid into a cell at low cost, simply and safely, efficiently and with low toxicity without requiring a special device. It is to provide a method for intracellular introduction and a compound carrier agent therefor.

  As a result of intensive studies to achieve the above object, the inventors of the present invention have the following formula (I), which is a known cationic amphiphile:

(Wherein, m represents 1 or 2, and n represents an integer of 2 to 6, respectively), or the following formula (II):

Surprisingly, when a compound represented by the formula (wherein p represents an integer of 14 to 18) is used as a carrier, compared to various conventionally known cationic lipid carriers, relatively low molecular nucleic acids and the like It has been found that the introduction efficiency of low molecular weight compounds into cells is high and the cytotoxicity is low. As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
(1) The following formula (I):

(In the formula, m represents 1 or 2, and n represents an integer of 2 to 6, respectively)
A carrier agent for intracellular introduction of a compound having a molecular weight of about 2 million or less, comprising a compound represented by:
(2) The agent according to (1) above, wherein n is an integer of 2 to 4;
(3) The agent according to the above (1), wherein n is 2.
(4) The agent according to any one of (1) to (3) above, wherein the compound represented by formula (I) is in the form of a salt with halogen;
(5) The agent according to any one of (1) to (4) above, wherein the molecular weight of the compound is about 700,000 or less;
(6) The agent according to any one of (1) to (5), wherein the compound is negatively charged;
(7) The agent according to any one of (1) to (6), wherein the compound is a nucleic acid;
(8) The agent according to any one of (1) to (7) above, wherein the compound has disease prevention / treatment activity;
(9) The agent according to any one of (1) to (8) above, wherein the compound is capable of controlling the expression of the target gene;
(10) The agent according to any one of (1) to (9) above, wherein the compound is any nucleic acid selected from the group consisting of siRNA, miRNA, antisense oligonucleotide, ribozyme and decoy oligonucleotide;
(11) A complex of the agent according to any one of (1) to (10) above and a compound having a molecular weight of about 2 million or less;
(12) The complex according to (11) above, wherein the molecular weight of the compound is about 700,000 or less;
(13) The complex according to (11) or (12) above, wherein the compound is negatively charged;
(14) The complex according to any one of (11) to (13), wherein the compound is a nucleic acid;
(15) The complex according to any one of (11) to (14) above, wherein the compound has disease prevention / treatment activity;
(16) The complex according to any one of (11) to (15) above, wherein the compound is capable of controlling the expression of the target gene;
(17) The complex according to any one of (11) to (16) above, wherein the compound is any nucleic acid selected from the group consisting of siRNA, miRNA, antisense oligonucleotide, ribozyme and decoy oligonucleotide;
(18) A method for introducing a compound into the cell, comprising bringing the complex according to any one of (11) to (17) above into contact with the cell;
(19) The method according to (18) above, wherein the cell is an animal or plant cell;
(20) The method according to (18) above, wherein the cell is a mammalian cell; and (21) administering the complex according to any of (11) to (17) above to a human or non-human subject. A method for introducing a compound into a cell in a living body of the subject,
I will provide a.

Furthermore, the present invention provides
(22) Formula (II) below:

(Wherein p represents an integer of 14 to 18)
A carrier agent for intracellular introduction of a compound having a molecular weight of about 2 million or less, comprising a compound represented by:
(23) The agent according to the above (22), wherein p is an integer of 14 to 16;
(24) A complex of the agent according to (22) or (23) above and a compound having a molecular weight of about 2 million or less;
(25) A method for introducing a compound into the cell, which comprises bringing the complex according to (24) above into contact with the cell;
(26) A method for introducing a compound into a cell in a living body of a subject, comprising administering the complex according to (24) to a human or a non-human subject;
(27) The compounds represented by formula (I) or (II) are provided in the form of aggregates organized in an aqueous solvent, (1) to (10) above, (22) or (23 ) Described agent;
(28) The agent according to (27) above, wherein the aqueous solvent can be heat-treated; and (29) the agent according to (28) above, wherein the aqueous solvent is a solution containing sodium chloride or potassium chloride;
I will provide a.

  The cationic lipid carrier in the present invention is a cell having a relatively high efficiency for relatively low molecular weight compounds such as siRNA and other low molecular nucleic acids, compared to cationic lipids used in conventional nucleic acid introduction reagents. It has an advantageous effect that it can be introduced into the cell and has low cytotoxicity. In addition, the cationic lipid molecule can be synthesized at a relatively low cost than the cationic lipid in the conventional nucleic acid introduction reagent. Furthermore, since the cationic lipid molecule is excellent in thermal stability, it can be handled at room temperature or higher, which is advantageous in terms of operability and transportation cost. In addition, it is advantageous as a carrier for introducing a compound into a cell from the viewpoint that the quality is stable due to being hardly subjected to chemical modification by oxidation or ultraviolet rays. Furthermore, the cationic lipid molecule forms a preferable molecular assembly not only in a solution unsuitable for heat treatment such as a commonly used culture solution but also in a heat treatable solution such as NaCl solution. It is difficult to remove (inactivate) impurities that impair the stability of the compound to be introduced, such as enzymes such as RNase, DNase, protease, and peptidase. The carrier agent for introduction into cells can be prepared after sterilization and virus inactivation treatment for preventing infection with the target bacteria and viruses.

  The carrier agent for intracellular introduction of the compound in the present invention (hereinafter sometimes abbreviated as “the carrier agent of the present invention”) has the following formula (I) as the main component [that is, the carrier of the compound (carrier)]:

(In the formula, m represents 1 or 2, and n represents an integer of 2 to 6, respectively)
The cationic lipid molecule represented by these is contained.
The compound introduced into the cells by the carrier agent of the present invention is not particularly limited as long as it has a molecular weight of about 2 million or less.
Cationic lipid molecules used for gene transfer generally interact with a compound (preferably a negatively charged compound) to form a stable complex by forming a stable complex (preferably electrostatic interaction). The cationic lipid molecules used in the present invention are particularly low molecular nucleic acids and the like, while suppressing the degradation of the protein (eg, by nuclease and peptidase) and facilitating delivery to the negatively charged cell surface. It has the advantage of being excellent in the efficiency of introduction of a relatively low molecular weight compound into cells and having low cytotoxicity.

The cationic lipid molecule used in the carrier agent of the present invention is an amphiphilic synthetic molecule having a hydrophilic part and a hydrophobic part in the same molecule. In the present specification, the number of carbon atoms in the two alkyl chains of the hydrophobic part of the cationic lipid molecule is 12, the number of carbon atoms in the acyl group of the hydrophilic part is n, and the part connecting the hydrophobic part and the hydrophilic part is , M has an aspartic acid skeleton, m has a glutamic acid skeleton when m is 2, and hereinafter, when m is 1, 12 Aspn or 12An (n is an integer of 2 to 6) and m is 2 In some cases, 12Glun or 12Gn (n is an integer of 2 to 6) may be abbreviated.
Preferably, the cationic lipid used in the carrier agent of the present invention is a compound in which n is an integer of 2 to 4 in Formula (I) (ie, 12A2 to 12A4 or 12G2 to 12G4), particularly preferably n Is an integer of 2, ie 12A2 or 12G2.

  In particular, in the cationic lipid molecule used in the carrier agent of the present invention, the compound in which m is 2 is excellent in water solubility and easy to handle.

  The compound represented by the formula (I) can be blended in the carrier agent of the present invention in the form of a salt with halogen, for example. Preferably it is provided as a chloride or bromide salt.

  The compound represented by the formula (I) can be produced by any method known per se, such as “Journal of the American Chemical Society, vol. 102, p6642 (1980)”, “Bulletin of the Chemical Society”. of Japan, vol. 64, p3677 (1991) ”and“ Biochemistry and Molecular Biology International, vol. 34, p915 (1994) ”, but are not limited thereto.

  The carrier agent of the present invention may contain any one of the above cationic lipid molecules alone, or may contain two or more kinds in combination. Alternatively, the carrier agent may be an amphipathic molecule other than the cationic lipid molecule (for example, phosphatidylinositol, etc.) as long as it does not impair the advantages of the present invention such as the intracellular introduction efficiency of the compound and low cytotoxicity. Phosphatidylserine, phosphatidylethanolamine, phospholipids derived from biological membranes such as phosphatidylcholine), surfactants (CHAPS, sodium cholate, octylglucoside, ND-gluco-N-methylalkanamides, etc.), polyethylene glycol, Glycolipids, peptide lipids, proteins and the like may further be contained.

The carrier agent of the present invention is prepared by dispersing one or more of the above cationic lipid molecules, or other amphiphilic molecules in an appropriate dispersion medium, for example, an aqueous solvent, and organizing as necessary. It can be prepared in a state where a molecular assembly is formed by performing an operation for inducing. Here, “organization” means that an amphiphilic molecule containing a cationic lipid is assembled through a non-covalent bond such as a hydrophobic bond to form an aggregate. Examples of the “operation for inducing organization” include, for example, ultrasonic treatment, heating, vortex, ether injection method, French press method, cholic acid method, Ca ²⁺ fusion method, freeze-thaw method, reverse phase evaporation method. [The details of each method are described in, for example, “Chapter 2 Preparation of Liposomes” of “Liposome” (Nanedo, published in 1988) edited by Nojima, Sunamoto and Inoue, for example. (Described in Sunamoto, Iwamoto, etc.)], but not limited thereto. In addition, under certain conditions, the amphiphilic molecules containing the cationic lipids gather and gather autonomously in an aqueous solvent without performing the above-described artificially induced manipulation. The body can also be formed (self-organized).
As an aggregate formed by organization, a bilayer membrane, a liposome, a multivesicle, a string-like aggregate, a disk-like aggregate, a lamellar-like aggregate formed by hydrophobic bonding of the hydrophobic parts of amphiphilic molecules, Rod aggregates and the like and mixtures thereof are included. The aggregate obtained by self-organization is usually a mixture of the above-mentioned various forms. However, an assembly having a single form can be obtained by performing the above-described operation for inducing the organization under a certain condition. It is also possible to form.

In a preferred embodiment, the cationic lipid molecule used in the preparation of the carrier agent of the present invention is a solid, more preferably a powder. The dispersion medium for dispersing the cationic lipid molecules is not particularly limited. For example, water (deionized water, etc.), physiological saline, phosphate buffered saline (PBS), and those skilled in the art can perform normal cell culture. An aqueous solvent such as a medium to be used (for example, RPMI 1640, DMEM, HAM F-12, Eagle medium, etc.) can be mentioned. It is preferable that the aqueous solvent does not contain protein components such as serum, but by removing the protein components in advance by treatment with polylysine or the like, amphipathic molecules including cationic lipids are organized or subsequently introduced into cells. Inhibition of complex formation between the compound and the cationic lipid molecule aggregate can also be prevented. In addition, when the compound to be introduced into the cell is a nucleic acid such as RNA or DNA, or a peptide compound such as an oligopeptide or protein, a nucleic acid degrading enzyme such as RNase or DNase, or a protein (peptide) degrading such as peptidase or protease. Since the stability of the introduced compound is reduced due to the mixing of the enzyme, the aqueous solvent is preferably subjected to a heat treatment in order to deactivate the enzyme before dispersing the cationic lipid molecules. The heat treatment can be performed, for example, at about 50 to about 100 ° C. for about 5 minutes to about 3 hours, but is not limited thereto. Therefore, the aqueous solvent is preferably one that can be subjected to the heat treatment. Conventionally, it is often impossible to remove enzymes such as RNase in various culture solutions used for introduction of nucleic acids into cells, but cationic lipid molecules in the present invention include compounds such as NaCl and potassium chloride. Even when dispersed in the contained aqueous solution, high introduction efficiency is exhibited in the subsequent intracellular introduction operation. Accordingly, when a compound such as a nucleic acid or a peptide compound is introduced into cells, an aqueous solution containing the above compound is preferably exemplified as the aqueous solvent.
Although pH of an aqueous solvent is not specifically limited, It is preferable that it is the range of pH 4-10, More preferably, it is the range of pH 7-8.

As a preferred embodiment, the preparation of cationic liposomes by (1) ultrasonic treatment and (2) heat treatment will be described more specifically below.
(1) Ultrasonic treatment method First, the cationic lipid molecules (for example, 12G2, 12A2 and the like) are dissolved in an organic solvent (for example, chloroform and the like), placed in a container such as an eggplant type flask, and the like using a rotary evaporator or the like. The solvent is removed under reduced pressure to form a lipid film on the wall of the container. An aqueous solvent (for example, phosphate buffer (pH 7.0) etc.) is added to this and swollen with shaking, and the thin film is peeled off using, for example, a vortex mixer to obtain a suspension of multilamellar liposomes. . Here, the swelling operation is preferably performed at a temperature equal to or higher than the phase transition temperature of the cationic lipid (Tc; for example, Tc of 12G2 is 4 ° C. and Tc of 12A2 is 6.7 ° C.). In addition, in order to remove decomposed lipids or the like, gel filtration can be performed using a Sephadex 2B, 4B or G-50 column.

  The suspension of multilamellar liposomes obtained as described above is high at a temperature equal to or higher than the Tc of the cationic lipid on an ice bath or water bath using an ultrasonic treatment device (probe type, bathtub type, etc.). By applying ultrasonic waves with an output (for example, about 100 to about 200 W) for about 1 to about 2 minutes (for example, 1 minute irradiation, repeating a cycle of 30 seconds intervals about 2 to 4 times, etc.) Unilamellar liposomes can be prepared.

  In the cationic lipid molecule of the present invention, simply taking the powder in a tube, adding MiliQ water or the like (so that the final concentration is about 20 mM), and performing ultrasonic treatment similar to the above, Cationic liposomes can be easily prepared.

(2) Heat treatment method Take an appropriate amount of powder of the above-mentioned cationic lipid molecules (for example, 12G2, 12A2, etc.) into a tube, add MiliQ water, etc. (so that the final concentration is about 20 mM), and about 90 ° C. Cationic liposomes can be prepared by heating at about 15 minutes.

The concentration of the cationic lipid molecule in the obtained carrier agent can be appropriately set in consideration of the kind of the cationic amphiphilic molecule to be used, etc., but is usually 1 to 200 mM, preferably 1 to 100 mM, more preferably 1. -50 mM, more preferably 5-50 mM, most preferably 10-30 mM.
If the concentration is too low, a sufficient amount of cationic amphiphilic molecule aggregates will not be formed, and if the concentration is too high, cationic amphiphilic molecules may precipitate.

  The carrier agent of the present invention is a suitable additive in addition to the amphiphilic molecule that forms a molecular assembly serving as a carrier of the compound within a range that does not impair the advantages of the present invention such as the efficiency of intracellular introduction of the compound and low cytotoxicity. May be included. When the carrier agent of the present invention is used for the purpose of introducing the compound into cells in the living body, the additive must be pharmaceutically acceptable. For example, various pharmaceutical additives blended in conventionally known liposome preparations can be used.

  The carrier agent of the present invention comprising the cationic lipid molecule, which can be obtained as described above, is useful as a drug for efficiently introducing a compound into cells with low toxicity. The compound that can be introduced into cells by the carrier agent of the present invention is not particularly limited as long as the molecular weight is about 2 million or less. For example, nucleic acids, peptides, lipids, sugars, lipids, sugars, physiologically active substances, drugs (Doxorubicin (Antineoplastic), Daunorubicin (Antineoplastic), Vincristine (Antineoplastic), Vinblastine (Antineoplastic), Idarubicin (Antineoplastic), Dibucaine (Local anesthetic), Propranolol (β-blocker), Quidinidine ( Arrhythmia drug), Dopamine (cardiac / pressor drug), Imipramine (anti-depressant), Diphenhydramine (anti-histamine), Quinine (anti-malarial), Chloroquine (anti-malarial), Diclofenac (anti-inflammatory), etc.), Moisturizers for cosmetics (mannitol, etc.), other synthetic or natural compounds such as other synthetic or natural compounds, and the like, preferably a compound having a molecular weight of about 1,000,000 or less, more preferably about 700,000 or less. It is.

  In addition, since the carrier agent of the present invention contains the cationic lipid molecule as a main component, in order to form a stable complex with the cationic lipid molecule aggregate, the compound is negatively charged. Preferably it is. Accordingly, preferred examples of the compound that can be introduced into cells by the carrier agent of the present invention include polyanions of nucleic acids and peptides.

  Particularly preferred compounds that can be introduced into cells by the carrier agent of the present invention are nucleic acids. The nucleic acid is not particularly limited as long as it has a molecular weight of about 2 million or less, and may be any DNA, RNA, DNA-RNA chimeric nucleic acid, DNA / RNA hybrid or the like. The nucleic acid can be any one of 1 to 3 strands, but is preferably a single strand or a double strand. Nucleic acids may be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers having a non-nucleotide backbone (eg, commercially available peptide nucleic acids (PNA), etc.) or other oligomers containing special linkages (However, the oligomer may contain nucleotides having a configuration that allows base pairing or base attachment as found in DNA or RNA). Furthermore, those with known modifications, such as those with labels known in the art, capped, methylated, one or more natural nucleotides substituted with analogs, Intramolecular nucleotide modifications, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg, phosphorothioates, phosphoro Having side chain groups such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg monosaccharides, etc.) Possessing an intercurrent compound (eg, acridine, psoralen, etc.) Those containing chelating compounds (eg metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg α-anomeric type) Nucleic acid etc.).

  Preferably, the size of the small molecule nucleic acid (when double-stranded) is about 3000 bp or less, more preferably about 1 to about 2000 bp, more preferably about 1 to about 1000 bp, and particularly preferably about 5 to about 1000 bp.

The nucleic acid may be either naturally occurring or synthesized, but if it has a size of about 100 bp or less, the phosphotriethyl method, the phosphodiester method, etc. can be used to make use of a commonly used nucleic acid automatic synthesizer. It is possible to synthesize.
The nucleic acid used in the present invention is not particularly limited, but is preferably purified by a method commonly used by those skilled in the art.

  The carrier agent of the present invention is used to introduce a compound into cells in a living body, for example, a so-called gene for the purpose of preventing and / or treating a disease (hereinafter abbreviated as “prevention / treatment”). The use in the in vivo administration of the prophylactic / therapeutic compound including a treatment is mentioned. Accordingly, in a preferred embodiment of the present invention, the compound introduced into cells by the carrier agent of the present invention has preventive / therapeutic activity for a certain disease. Examples of such compounds include nucleic acids, peptides, lipids, sugars, physiologically active substances, drugs, and other natural or synthetic compounds.

  As described above, gene therapy can be broadly divided into those intended to compensate for the missing genetic information and those intended to control the expression of the causative gene of the disease. In addition to the base sequence encoding the deleted protein, the nucleic acid to be used includes a promoter sequence that can function in the introduced cell, and a base sequence such as a polyadenylation signal, a selection marker gene, and a replication origin (ori) as necessary. Therefore, the intracellular introduction (gene therapy) of a nucleic acid by the carrier agent of the present invention is substantially aimed at controlling the expression of the latter disease-causing gene. Therefore, the compound introduced into cells by the carrier agent of the present invention is preferably one that can control the expression of the target gene (disease gene).

  When the compound capable of controlling the expression of the target gene is a low-molecular nucleic acid, examples of the low-molecular nucleic acid include siRNA, miRNA, antisense oligonucleotide, ribozyme, decoy oligonucleotide (for example, a transcription factor or transcription repressor is recognized) And an oligonucleotide containing a base sequence that can be bound to each other.

  When the compound capable of controlling the expression of the target gene is a peptide or protein, the peptide / protein, for example, binds to the target gene and controls transcription of the gene, or is added to the mRNA or initial transcript of the target gene. Peptides / proteins that bind to control translation into proteins, peptide ligands that can enhance signals from receptors that control expression of target genes, or antagonist-like peptides / proteins that can block the signals It is done.

  Alternatively, a compound having disease prevention / treatment activity may be one that can control the activity of a disease-causing protein. Such examples include peptides / proteins that are ligands of the target receptor protein, non-peptide compounds (eg fatty acids, steroid hormones, etc.), various natural or synthetic compounds having agonist or antagonist activity, kinases Although the peptide etc. which mimic the partial amino acid sequence of the phosphorylation site are mentioned, it is not limited to them.

The present invention also provides a complex of the carrier agent of the present invention and a compound to be introduced into a cell, preferably a compound as already mentioned herein.
In order to use the amphipathic molecular assembly containing the cationic lipid molecule for introduction of a compound into a cell, the molecular assembly and the introduced compound are brought into contact with each other by bringing the molecular assembly into contact with the introduced compound. (Hereinafter sometimes referred to simply as “complex”). The complex may be formed by any interaction as long as the complex exists stably and can suppress degradation of the introduced compound (nucleic acid, peptide, etc.) by, for example, nuclease or peptidase. Since the molecular assembly is cationic, in a preferred embodiment, the complex is electrostatically bound to an anionic (ie, negatively charged) compound (nucleic acid, peptide, etc.). It is formed through non-covalent bonds by interaction, but even positively charged compounds or neutrally charged compounds can be pre-bonded through other interactions or with negatively charged compounds. A complex can be formed.
The complex of the amphipathic molecular assembly containing the cationic lipid molecule and the introduced compound is obtained by mixing and incubating the aqueous solvent containing the aggregate and the introduced compound. The kind of the aqueous solvent is the same as described above.
In addition, the temperature during the incubation is preferably set in the same range as the temperature in the method for preparing the cationic amphiphilic molecule assembly.

The concentration of the cationic lipid in the mixed solution can be appropriately set in consideration of the kind of the cationic lipid molecule used and the like, but is usually 1 to 200 mM, preferably 1 to 100 mM, more preferably 1 to 50 mM. More preferably, it is in the range of 5-50 mM, most preferably 10-30 mM.
When the concentration is too low, a sufficient amount of a stable complex is not formed, and when the concentration is too high, a cationic amphiphilic molecular aggregate may precipitate.
The concentration of the introduced compound in the mixture can be appropriately set in consideration of the type and size (molecular weight) of the compound to be used, but when the compound is a nucleic acid, it is usually about 0.01 to about 100 ng / μL, preferably about 0.1 to about 25 ng / μL, more preferably about 0.5 to about 10 ng / μL, more preferably about 0.5 to about 5 ng / μL, and most preferably about 0.5 to about 2 ng / μL. is there.
In particular, when the nucleic acid is a very small one of about 20 to about 200 bp like siRNA, the concentration of the nucleic acid is usually 1 to 500 nM, preferably 20 to 400 nM, more preferably 20 to 300 nM, still more preferably 20 to 200 nM, most preferably in the range of 20-100 nM.

The incubation time after mixing the aqueous solvent containing the aggregate and the introduced compound can be appropriately set in consideration of conditions such as the type of reagent used, but is usually 0.5 to 100 minutes, preferably Is in the range of 0.5 to 60 minutes, more preferably 0.5 to 30 minutes, still more preferably 0.5 to 15 minutes, and most preferably 1 to 5 minutes.
If the incubation time is too short, the complex formation between the introduced compound and the cationic amphiphilic molecule aggregate will be insufficient, and if the incubation time is too long, the formed complex may become unstable. In either case, the introduction efficiency of the introduced compound is lowered.
A mixed solution containing a complex of a cationic amphiphilic molecular assembly used for introducing the introduced compound into the cell and the compound by the above process (hereinafter sometimes referred to as “complex-containing solution”) Obtainable.

Furthermore, by bringing the complex obtained in the above step into contact with the cell, the introduced compound contained in the complex can be introduced into the cell.
The type of the “cell” is not particularly limited, and prokaryotic and eukaryotic cells can be used, but eukaryotic cells are preferable. The type of eukaryote is not particularly limited, and examples thereof include mammals including humans (human, monkey, mouse, rat, hamster, cow, etc.), birds (chicken, ostrich, etc.), amphibians (frog, etc.), fish (zebra) Vertebrates such as fish and medaka), invertebrates such as insects (eg, moths, moths, and fruit flies), and microorganisms such as plants and yeasts. More preferably, the cells targeted by the present invention are animal or plant cells, more preferably mammalian cells.
The cell may be a cultured cell line containing cancer cells, a cell isolated from an individual or tissue, or a tissue or tissue piece cell. Further, the cells may be adherent cells or non-adherent cells.

The step of bringing the complex into contact with the cells will be described more specifically as follows.
That is, the cells are suspended in an appropriate medium several days before contact with the complex and cultured under appropriate conditions. Upon contact with the complex, the cell may or may not be in the growth phase.

  The culture solution at the time of the contact may be a serum-containing medium or a serum-free medium, but the serum concentration in the medium is preferably 20% or less, preferably 10% or less. This is because if the medium contains excess protein such as serum, the contact between the complex and the cell may be inhibited.

The cell density at the time of the contact is not particularly limited and can be appropriately set in consideration of the cell type and the like. Usually, the cell density is 0.5 × 10 ^{5 to} 5 × 10 ⁵ cells / mL, preferably 0. 5 × 10 ⁵ to 4 × 10 ⁵ cells / mL, more preferably 0.5 × 10 ^{5 to} 3 × 10 ⁵ cells / mL, more preferably 1 × 10 ^{5 to} 3 × 10 ⁵ cells / mL, most preferably Is in the range of 1 × 10 ^{5 to} 2 × 10 ⁵ cells / mL.

  The above complex-containing solution is added to the medium containing the cells thus prepared. The addition amount of the complex-containing solution is not particularly limited and can be appropriately set in consideration of the number of cells and the like. Usually, 1 to 1000 μL, preferably 1 to 500 μL, more preferably 1 per 1 mL of the medium. It is -300 microliters, More preferably, it is 1-200 microliters, Most preferably, it is the range of 1-100 microliters.

After adding the complex-containing solution to the medium, the cells are cultured, and the temperature, humidity, CO ₂ concentration, etc. during the culture are appropriately set in consideration of the cell type. In the case of mammalian cells, the temperature is usually about 37 ° C., the humidity is about 95%, and the CO ₂ concentration is about 5%.
The culture time can also be appropriately set in consideration of conditions such as the type of cells used, but is usually 0 to 72 hours, preferably 1 to 48 hours, more preferably 1 to 36 hours, and still more preferably. It is in the range of 1-24 hours, most preferably 1-16 hours.
When the culture time is too short, the introduced compound is not sufficiently introduced into the cells, and when the culture time is too long, the cells may be weakened.

The low-molecular compound is introduced into the cells by the above culture, but preferably the medium is replaced with a fresh medium, or the fresh medium is added to the medium and the culture is further continued. If the cells are mammalian cells, the fresh medium preferably contains serum or nutrient factors.
The time for further culturing can be appropriately set in consideration of functions expected of the introduced low molecular weight compound, but the low molecular weight compound can control the expression of a target gene such as siRNA. In the case of a low molecular nucleic acid, it is usually in the range of 0 to 72 hours, preferably 1 to 48 hours, more preferably 1 to 36 hours, still more preferably 1 to 24 hours, and most preferably 1 to 16 hours.

Further, as described above, by using a complex of an amphipathic molecular assembly containing a cationic lipid and a low molecular weight compound, the complex can be used not only in a test tube (in vitro) but also in a living body (in vivo). It is possible to introduce the compound into the cell. That is, by administering the complex to a subject, the complex reaches / contacts the target cell, and the low molecular compound contained in the complex is introduced into the cell in vivo.
The subject to which the complex can be administered is not particularly limited. For example, mammals including humans (human, monkey, mouse, rat, hamster, cow, etc.), birds (chicken, ostrich, etc.), amphibians (frog, etc.) And vertebrates such as fish (eg zebrafish, medaka), invertebrates such as insects (eg, moths, moths, and fruit flies), and plants. Preferably, the subject of administration of the complex includes a human or other mammal.

In addition, the administration method of the complex is not particularly limited as long as the complex reaches / contacts the target cell and the introduced compound contained in the complex can be introduced into the cell. A known administration method (oral administration, parenteral administration (intravenous administration, intramuscular administration, local administration, transdermal administration, subcutaneous administration, intraperitoneal administration, etc.)) Can be appropriately selected.
The dose of the complex is not particularly limited as long as the introduction of the low molecular weight compound into the cell can be achieved, taking into consideration the type of administration target, the administration method, the type of the introduced compound, the type and site of the target cell, etc. In the case of oral administration, for example, generally in humans (as 60 kg), the single dose is about 0.001 mg to 10,000 mg as a complex. When administered parenterally (for example, intravenous administration), generally, for example, in humans (as 60 kg), the single dose is about 0.0001 mg to 3000 mg as a complex. In the case of other animals, an amount converted per 60 kg can be administered.

  The carrier agent of the present invention can also be provided as a kit for introducing a compound into cells. The kit may further contain any reagent that can be used in the intracellular introduction method of the compound using the carrier agent of the present invention (for example, the above-mentioned aqueous solvent, instructions describing the preparation protocol, reaction container, etc.). I can do it. By using this kit, it is possible to easily introduce a desired compound into cells according to the method described above.

  As another cationic lipid molecule that can be used in the carrier agent of the present invention, the following formula (II):

(Wherein, p represents an integer of 14 to 18). In the present specification, since the number of carbon atoms in the two alkyl chains of the hydrophobic portion of the cationic lipid molecule is p, hereinafter, it may be abbreviated as Cp.
Preferably, the cationic lipid used in the carrier agent of the present invention is a compound in which p is an integer of 14 to 18 in Formula (II) (ie, C14 to C18), and particularly preferably p is 14 to 16 A compound that is an integer of C14 to C16.

  The compound represented by the formula (II) can be blended in the carrier agent of the present invention, for example, in the form of a salt with halogen. Preferably it is provided as a chloride or bromide salt.

  The compound represented by the formula (II) can be produced by any method known per se, such as “Journal of the American Chemical Society, vol. 102, p6642 (1980)” and “Biochemistry and Molecular Biology International”. , vol. 34, p915 (1994) ", but is not limited thereto.

  A method for preparing a compound represented by formula (II) as a carrier agent of the present invention, a method for forming a complex with a compound introduced into a cell using the obtained carrier agent, and the resulting complex being a cell. As the method for introducing the compound and the various materials used in these methods, the same materials as those described above for the compound represented by formula (I) can be used.

  The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the scope of the present invention.

Examination of cationic lipid carrier suitable for introduction of siRNA into CHO cells
CHO-EGFP cells (CHO cells that constitutively express the fluorescent protein Enhanced Green Fluorescent Protein (EGFP); prepared by transfecting EGFP gene expression vectors into CHO cells (source: RIKEN) by conventional methods) 1-2 × 10 ⁵ cells / well were precultured in 24-well plates for 16 hours (in DMEM culture medium containing 10% FBS), and then replaced with 0.5 ml of serum-free culture medium at the time of introduction.
100 pM EGFP siRNA (source: NIPPON GENE) is mixed with 25 μl of serum-free medium, and 1.3 mM of various cationic lipids [12G2, 12G4, 12G6, 12G11, 14G2, 14G4 and 14G6, 12A2, 14A2 and 16A2, C14, C16 and C18, TMA (Chemical 7), PC14 (Chemical 8), 8AzC10 (Chemical 9) or 12GN3 (Biochemistry and Molecular Biology International, vol.34, p915 (1994), see FIG. 1)] 5 each μl was mixed with the above siRNA solution and incubated for 5 minutes to obtain a cationic lipid molecule assembly-siRNA complex. This was added to the above CHO-EGFP cells, incubated for 4 hours at 37 ° C. in a CO ₂ incubator (5% CO ₂ /95% air), and then replaced with a medium containing serum. The next day, the cells were observed and fluorescence was measured by flow cytometry. The results are shown in FIG. 1 and FIG. EGFP expression is shown as relative intensity with the fluorescence intensity in Control (siRNA non-introduced cells) as 100%. When 12G2, 12G4 and 12G6 (Fig. 1), or 12A2, C14, C16 and C18 (Fig. 2) are used as carriers, EGFP expression is remarkably suppressed and siRNA is introduced into cells at a high frequency. I understood that. In particular, 12G2 and 12A2 showed high cell transfer efficiency.

Comparative examination of 12G2 and 12A2 and commercially available nucleic acid introduction reagents In the same manner as in Example 1, 12G2 and 12A2 and various commercially available lipofection reagents [Lipofectin, Lipofectamine, Lipofectamine2000, Oligofectamine (above, Invitrogen), DoTAP (Biontex), The siRNA introduction efficiency was compared with FuGENE6 (Roche), CLONfectin (Clontech), JetSI (PolyPlus-transfection), TransIT-TKO (Mirus), siFECT (IC-VEC), and siFECTOR (B-Bridge International Inc.). The results are shown in FIG. 12G2 and 12A2 showed high siRNA cell transfer efficiency comparable to JetSI, TransIT-TKO and siFECT.

Introducing siRNA into cultured human cells using 12G2 and 12A2 1-2 x 10 ⁵ cells / well of normal human hepatocytes (HC cells) or human uterine cancer cells (HeLa cells) in a 24-well plate for 16 hours (10% FBS-containing FD medium (HC cells), 10% FBS-containing DMEM medium (HeLa cells)) and then replaced with 0.5 ml of serum-free medium at the time of introduction.
Mix 100 pM Cy-3 fluorescently labeled siRNA (source: B-Bridge) in 25 μl serum-free medium, mix 1.3 mM 12Glu2 or 12Asp2 (5 μl) in the above siRNA solution and incubate for 5 minutes. Thus, a cationic lipid molecule assembly-siRNA complex was obtained. This was added to the HC cells or HeLa cells, incubated for 4 hours at 37 ° C. in a CO ₂ incubator (5% CO ₂ /95% air), and then replaced with a medium containing serum. The next day, the cells were observed using a fluorescence microscope and a phase contrast microscope. The results are shown in FIGS. In both 12Glu2 and 12Asp2, siRNA introduction efficiency was almost 100% in both HC cells and HeLa cells.

In vivo siRNA transfer into mouse tissues using 12G2 and 12A2
2 nM Cy-3 fluorescently labeled siRNA was mixed with 200 μl NaCl aqueous solution. Each 50 μl of 1.3 mM 12Glu2 or 12Asp2 was mixed with the above aqueous solution and incubated for 5 minutes. This mixture was injected from the tail vein of Balb / c mice (female, 7-8 weeks old), 4 hours later (or 24 hours later), the mice were dissected, and the cells were examined using a fluorescence microscope and phase contrast microscope. Observed. The results are shown in FIGS. Cy-3 labeled siRNA positive cells were remarkably confirmed in the spleen (FIG. 8), liver (FIG. 9) and lung. Cy-3 labeled siRNA positive cells were also confirmed in the heart (FIG. 10), kidney, and brain tissue.

Cytotoxicity test Cells (CHO cells, HC cells, HeLa cells) 1-2 × 10 ⁵ cells / well were pre-cultured in 24-well plates and then replaced with 0.5 ml of serum-free medium at the time of introduction.
Mix 100 pM Cy-3 fluorescently labeled siRNA in 25 μl of 150 mM NaCl solution, mix 1.3 mM 12Glu2 or 12Asp2 (5 μl) in the above siRNA solution and incubate for 5 minutes to obtain cationic lipid molecules. Aggregate-siRNA complexes were obtained. Only the complex or cationic lipid molecule aggregate is added to the CHO, HC or HeLa cells, incubated in a CO ₂ incubator (5% CO ₂ /95% air) at 37 ° C. for 4 hours, and then a medium containing serum Was replaced. After 4 hours, the medium was replaced with a serum-containing medium, and the culture was continued. The next day, cell death was evaluated by trypan blue staining. The results are shown in FIG. None of the cells into which 12Glu2 and 12Asp2 had been introduced showed a significant difference in cell viability from non-introduced cells.

Examination of mixed solution for siRNA / 12Glu2 complex formation
CHO-EGFP cells 1-2 × 10 ⁵ cells / well were pre-cultured in 24-well plates and then replaced with 0.5 ml of 10% FBS-DMEM culture medium at the time of introduction.
Mix 100 pM EGFP siRNA in 25 μl aqueous solution (DMEM medium, opti-MEM medium, 150 mM NaCl solution), mix 5 μl 1.3 mM 12Glu2 in the above siRNA solution, and incubate for 5 min. Sex lipid molecule assembly-siRNA complex was obtained. The complex was added to the cells and incubated at 37 ° C. in a CO ₂ incubator (5% CO ₂ /95% atmosphere). The next day, the cells were observed and fluorescence was measured by flow cytometry. The results are shown in FIG. Even when an aqueous NaCl solution was used as the aqueous solvent, the introduction efficiency was the same as when the culture medium was used.

Intracellular introduction of proteins and other low molecular weight compounds As introduction compounds, (1) fluorescent compound Sulforhodamine 101 (Molecular Probes; molecular weight 606.71) and (2) Albumin-Sulforhodamine 101 (Sigma; molecular weight 65-70 kDa; albumin (1 ) And a fluorescent compound).
(1) Introduction of Sulforhodamine 101 Mouse neural progenitor cells (cells isolated and cultured according to conventional methods from Balb / C mouse fetal central nerve) 1-2 x 10 ⁵ cells / well were pre-cultured in a 24-well plate, At the time of introduction, the medium was replaced with a serum-free FD medium supplemented with 0.5 ml of nutrient factors.
0.5 μg of Sulforhodamine 101 is mixed with 25 μl of 150 mM NaCl solution, 3.5 μl of 1.3 mM 12Glu2 is mixed with the above solution and incubated for 5 minutes, and then the cationic lipid molecule assembly-Sulforhodamine 101 complex Got. The complex was added to the cells and incubated for 1 hour at 37 ° C. in a CO ₂ incubator (5% CO ₂ /95% atmosphere). After 1-2 hours, the cells were observed with a fluorescence microscope. The results are shown in FIG.

(2) Introduction of Albumin-Sulforhodamine 101
CHO cells were precultured at 1-2 × 10 ⁵ cells / well in a 24-well plate and then replaced with 0.5 ml of 10% FBS-DMEM medium at the time of introduction.
Mix 1.0 μg of Albumin-Sulforhodamine 101 in 25 μl of 150 mM NaCl solution, mix 7 μl of 1.3 mM 12Glu2 in the above solution, incubate for 5 minutes, and then add cationic lipid molecule assembly-Albumin- Sulforhodamine 101 complex was obtained. The complex was added to the cells and incubated at 37 ° C. in a CO ₂ incubator (5% CO ₂ /95% atmosphere). The next day, the cells were observed with a fluorescence microscope. The results are shown in FIG.
In both cases (1) and (2), almost 100% of positive cells were confirmed.

  The cationic lipid carrier used in the carrier agent of the present invention can efficiently introduce low molecular weight compounds including low molecular weight nucleic acids such as siRNA into cells and has low cytotoxicity. Cationic lipid molecules are relatively inexpensive and difficult to undergo chemical denaturation, and thus are useful as high-performance low-molecular-weight compound intracellular introduction reagents for research and medicine.

It is a figure which shows siRNA introduction | transduction efficiency to the CHO cell at the time of using various cationic lipids as a carrier. The vertical axis shows the relative expression (%) of EGFP. It is a figure which shows siRNA introduction | transduction efficiency to the CHO cell at the time of using various cationic lipids as a carrier. The vertical axis shows the relative expression (%) of EGFP. It is a figure which shows the comparison of siRNA introduction | transduction efficiency to CHO cell of 12G2 and 12A2, and various commercially available cationic lipids. The vertical axis shows the relative expression (%) of EGFP. It is the fluorescence micrograph (left) and phase-contrast micrograph (right) in the HC cell which introduce | transduced Cy-3 fluorescence label | marker siRNA using 12Glu2 as a cationic lipid carrier. It is the fluorescence micrograph (left) and phase-contrast micrograph (right) in the HC cell which introduce | transduced Cy-3 fluorescence label | marker siRNA using 12Asp2 as a cationic lipid carrier. It is the fluorescence micrograph (left) and phase-contrast micrograph (right) in the HeLa cell which introduce | transduced Cy-3 fluorescence label | marker siRNA using 12Glu2 as a cationic lipid carrier. It is the fluorescence micrograph (left) and phase-contrast micrograph (right) in HeLa cell which introduce | transduced Cy-3 fluorescence label | marker siRNA using 12Asp2 as a cationic lipid carrier. It is the fluorescence micrograph (left) and phase-contrast micrograph (right) in the mouse | mouth spleen which introduce | transduced Cy-3 fluorescence labeled siRNA in vivo using 12Glu2 as a cationic lipid carrier. It is the fluorescence micrograph (left) and phase-contrast micrograph (right) in the mouse liver which introduce | transduced Cy-3 fluorescence label | marker siRNA in vivo using 12Glu2 as a cationic lipid carrier. It is the fluorescence micrograph (left) and phase-contrast micrograph (right) in the mouse | mouth heart which introduce | transduced Cy-3 fluorescence labeled siRNA in vivo using 12Glu2 as a cationic lipid carrier. It is a figure which shows the influence which it has on the cell viability of the intracellular introduction | transduction of 12Glu2 and 12Asp2. The vertical axis represents the cell viability (%). It is a figure which shows the influence which it has on siRNA introduction | transduction efficiency of various aqueous solvents. The vertical axis shows the relative expression (%) of EGFP. It is the fluorescence micrograph (left) and phase-contrast micrograph (right) in the mouse | mouth neural progenitor cell which introduce | transduced Sulforhodamine 101 by using 12Glu2 as a cationic lipid carrier. It is the fluorescence micrograph (left) and phase-contrast micrograph (right) in the CHO cell which introduce | transduced Albumin-Sulforhodamine 101 by using 12Glu2 as a cationic lipid carrier.

Claims (21)

The following formula (I):

(In the formula, m represents 1 or 2, and n represents an integer of 2 to 6, respectively)
A carrier agent for intracellular introduction of a compound having a molecular weight of about 2 million or less, comprising a compound represented by the formula:   The agent according to claim 1, wherein n is an integer of 2 to 4.   The agent according to claim 1, wherein n is 2.   The agent according to any one of claims 1 to 3, wherein the compound represented by the formula (I) is in the form of a salt with halogen.   The agent in any one of Claims 1-4 whose molecular weight of a compound is about 700,000 or less.   The agent according to any one of claims 1 to 5, wherein the compound is negatively charged.   The agent according to any one of claims 1 to 6, wherein the compound is a nucleic acid. The agent according to any one of claims 1 to 7, wherein the compound has a disease prevention / treatment activity.   The agent according to any one of claims 1 to 8, wherein the compound is capable of controlling the expression of a target gene.   The agent according to any one of claims 1 to 9, wherein the compound is any nucleic acid selected from the group consisting of siRNA, miRNA, antisense oligonucleotide, ribozyme and decoy oligonucleotide.   A complex of the agent according to any one of claims 1 to 10 and a compound having a molecular weight of about 2 million or less.   The complex of claim 11, wherein the molecular weight of the compound is about 700,000 or less.   13. A complex according to claim 11 or 12, wherein the compound is negatively charged.   The complex according to any one of claims 11 to 13, wherein the compound is a nucleic acid.   The complex according to any one of claims 11 to 14, wherein the compound has a disease prevention / treatment activity.   The complex according to any one of claims 11 to 15, wherein the compound is capable of controlling the expression of a target gene.   The complex according to any one of claims 11 to 16, wherein the compound is any nucleic acid selected from the group consisting of siRNA, miRNA, antisense oligonucleotide, ribozyme and decoy oligonucleotide.   A method for introducing a compound into a cell, comprising bringing the complex according to any one of claims 11 to 17 into contact with the cell.   The method according to claim 18, wherein the cell is an animal or plant cell.   The method of claim 18, wherein the cell is a mammalian cell.   A method for introducing a compound into a cell in a living body of a subject, comprising administering the complex according to any one of claims 11 to 17 to a human or a non-human subject.