JP5382682B2 - Drug delivery complex - Google Patents

Description

  The present invention relates to novel drug delivery complexes. More specifically, the present invention relates to a drug delivery complex comprising an anionic molecule encapsulating a complex of a drug and a cationic molecule.

  Since the surface of cells is negatively charged, methods for promoting absorption (uptake) of pharmaceuticals using positively charged cationic molecules have been actively studied. Various cationic molecules have been studied for gene transfer using non-viral vectors, and cationic polymers and lipids having high gene transfer efficiency have been reported. However, many of them have high cytotoxicity and bind nonspecifically to blood and various organs, thereby causing side effects such as hemagglutination and difficult gene targeting. For these problems, a method of modifying with a hydrophilic polymer such as polyethylene glycol (PEG) was considered, but the retention in the blood was improved, but the contact with cells was reduced due to its steric hindrance, There was a major drawback that sufficient gene expression could not be obtained. Although attempts have been made to improve the delivery efficiency to cells by modifying the PEG chain with antibodies and ligands for cell surface receptors, it is predicted that it will be difficult to develop as a pharmaceutical because of the complexity of the components Is done.

  Attempts have been made to overcome the disadvantages of such cationic molecules by reducing the positive charge on the surface using anionic molecules. For example, Non-Patent Document 1 discloses that a complex of polyethyleneimine (hereinafter sometimes abbreviated as PEI) and DNA is coated with a small amount of alginate to reduce the positive charge on the surface, thereby causing hemagglutination or It is described that the cytotoxicity is reduced.

On the other hand, a nucleic acid introduction reagent using only an anionic molecule has also been reported. For example, Patent Document 1 discloses that a non-encapsulated preparation containing a nucleic acid and polyglutamic acid is locally administered and is electroporated into muscle cells. It is described that it was introduced.
However, a drug delivery system (DDS) that has no side effects such as cytotoxicity and hemagglutination, can efficiently target a drug such as nucleic acid to a desired organ, and can provide sufficient gene expression has not yet been developed. .
International Publication No. 01/066149 Pamphlet Jiang, G. et al., Yao Xue Xue Bao, 41 (5): 439-45 (2006)

  An object of the present invention is to provide a drug delivery system that is less damaging to a living body and that can selectively deliver a drug to cells at a target site.

  As a result of intensive studies to solve the above problems, the present inventors have determined that a complex of a nucleic acid and a cationic molecule is γ-polyglutamic acid (hereinafter sometimes abbreviated as γ-PGA), chondroitin sulfate, Coating with any anionic molecule of alginic acid to form a complex that is substantially uncharged or has a negative surface charge results in low cytotoxicity, without causing hemagglutination, It has been found that cells exhibit transfection efficiency comparable to nucleic acid-cationic molecule complexes, and that nucleic acids can be efficiently delivered to the spleen by systemic administration in vivo. Similar results were obtained when the drug-cationic molecule complex was in the form of micelles or liposomes, so that not only negatively charged drugs such as nucleic acids but also positively charged or electrically neutral lipid solubility Alternatively, it was confirmed that this method can be applied as a delivery system for any water-soluble drug.

  Since the drug delivery complex of the present invention is substantially uncharged or has a negative surface charge, it does not cause hemagglutination, has low cytotoxicity, and is excellent in intracellular uptake efficiency. Can be delivered into the cell. The selection of anionic molecules also allows selective drug delivery to the spleen by systemic administration such as intravenous administration.

That is, the present invention provides the following.
[1] A drug delivery complex containing a complex of a drug and a cationic molecule and an anionic molecule encapsulating the complex, and having a substantially uncharged or negative surface charge, the anionic molecule A drug delivery complex wherein is selected from the group consisting of γ-polyglutamic acid, chondroitin sulfate, alginic acid and salts thereof.
[2] The complex of the drug and the cationic molecule is selected from the group consisting of a complex by self-assembly of the drug and the cationic molecule, a cationic micelle encapsulating the drug, and a cationic liposome encapsulating the drug. [1] The drug delivery complex according to [1].
[3] The drug delivery complex according to [1] or [2], wherein the drug is selected from the group consisting of nucleic acids, peptides, proteins, polysaccharides, and low molecular weight compounds.
[4] Any of [1] to [3], wherein the molar ratio of the functional group having a positive charge of the cationic molecule to the functional group having a negative charge of the anionic molecule is 3: 1 to 1: 4. A drug delivery complex according to 1.
[5] The drug delivery complex according to any one of [1] to [4], wherein the molecular weight of the anionic molecule is 100,000 or less.
[6] The drug delivery complex according to [5], wherein the anionic molecule is γ-polyglutamic acid or a salt thereof.
[7] The drug delivery complex according to any one of [1] to [6], which is for delivering a drug to the spleen.
[8] The drug delivery complex according to any one of [1] to [7], which can be administered systemically.
[9] A method for delivering a drug into a cell, comprising bringing the drug delivery complex according to any one of [1] to [6] into contact with the cell.
[10] A method for delivering a drug into a cell of an animal, comprising administering the drug delivery complex according to any one of [1] to [8] to a mammal.

  The present invention provides a complex (referred to herein as a “drug delivery complex”) for delivering a drug into a desired cell. The drug delivery complex of the present invention contains a complex of a drug and a cationic molecule and an anionic molecule enclosing it, and is characterized by having a substantially uncharged or negative surface charge.

  The anionic molecules used in the drug delivery complex of the present invention may be γ-polyglutamic acid (γ-PGA), chondroitin sulfate (hereinafter sometimes abbreviated as CS), alginic acid (hereinafter abbreviated as AGA). ) And their salts. Examples of γ-PGA or a salt thereof include compounds represented by the following formula (1).

(In the formula, R is a hydrogen atom, an alkali metal atom such as sodium, potassium, lithium, etc., or a tertiary compound such as trimethylamine, triethylamine, dimethylamine, diethylamine, triethanolamine, trimethanolamine, diethanolamine, dimethanolamine, ethanolamine, etc. An amine, or a quaternary amine such as tetramethylamine or tetraethylamine, and the Rs present in the molecule may be the same or different, and n is an integer of 40 or more.)

  Examples of CS or a salt thereof include compounds represented by the following formula (2).

(Wherein R ^{1 to} R ³ are each independently a hydrogen atom or a sulfonic acid group (provided that at least one of R ¹ or R ² is a sulfonic acid group), and R ^{4 to} R ⁶ are independently Hydrogen atoms, alkali metal atoms such as sodium, potassium, lithium, etc., trimethylamine, triethylamine, dimethylamine, diethylamine, triethanolamine, trimethanolamine, diethanolamine, dimethanolamine, ethanolamine and other tertiary amines, or tetramethyl It is a quaternary amine such as amine or tetraethylamine. The sulfonic acid groups present in the molecule are alkali metal atoms such as sodium, potassium and lithium, trimethylamine, triethylamine, dimethylamine, diethylamine, triethanolamine, and trimerene, respectively. Noruamin, diethanolamine, dimethanol amine, a tertiary amine, such as ethanolamine or tetramethylammonium amine, may be substituted with quaternary amines such as tetraethyl amine, n represents an integer of 10 or more.)

  Examples of AGA or a salt thereof include compounds represented by the following formula (3).

(Wherein R ^{1 to} R ⁴ are each independently an alkali metal atom such as a hydrogen atom, sodium, potassium, lithium, trimethylamine, triethylamine, dimethylamine, diethylamine, triethanolamine, trimethanolamine, diethanolamine, dimethanolamine) , A tertiary amine such as ethanolamine, or a quaternary amine such as tetramethylamine and tetraethylamine, m and n represent the total number of each block of the block copolymer, and the sum of m and n is 20 That's it.)

  Each of these anionic molecules can be prepared by a method known per se.

  When the anionic molecule is a polymer, that is, γ-PGA, CS, AGA or a salt thereof, the degree of polymerization is not particularly limited, but examples include those having a molecular weight of 5,000 or more, more preferably 10,000 or more. It is done. In addition, the anionic polymer has a molecular weight of, for example, 200,000 or less, preferably 150,000 or less, more preferably 100,000 or less, in that the drug can be stably encapsulated without being affected by the amount of the anionic molecule. .

  The cationic molecule used in the drug delivery complex of the present invention is not limited as long as it can form a complex by electrostatic interaction with the anionic molecule. For example, a cationic polymer [for example, polyethyleneimine (hereinafter referred to as polyethyleneimine) , May be abbreviated as PEI), polycationic polysaccharides such as chitin and chitosan, polylysine polypeptides such as polylysine, polyarginine, and protamine], or cationic lipids such as phosphatidylcholine (soybean phosphatidylcholine, egg yolk phosphatidylcholine, Distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, etc.), phosphatidylethanolamine (distearoylphosphatidylethanolamine, etc.), phosphatidylserine, phosphatidic acid, phosphatidylglycerol Phospholipids such as phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin, hydrogenated phospholipids, for example glyceroglycolipids such as sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride, for example Glycosphingolipids such as galactosyl cerebroside, lactosyl cerebroside, ganglioside, amino groups, alkylamino groups, dialkylamino groups, trialkylammonium groups, monoacyloxyalkyl-dialkylammonium groups, diacyloxyalkyl-monoalkylammonium groups, etc. And the like, but not limited to, a lipid having a quaternary ammonium group introduced therein.

  These cationic molecules can also be prepared by methods known per se.

  Any drug can be delivered into cells by the drug delivery complex of the present invention, such as nucleic acids, peptides, proteins, lipids, peptide lipids, sugars, low molecular weight compounds, other synthetic or natural compounds, etc. Is mentioned. When the intracellular delivery is intended to treat and / or prevent a disease, the drug has a therapeutic and / or prophylactic activity for the disease. For example, an antihypertensive agent, an antihypertensive agent, an antipsychotic disease Agent, analgesic agent, antidepressant, antidepressant, anxiolytic, sedative, hypnotic, antidepressant, opioid agonist, asthma treatment, anesthetic, antiarrhythmic agent, arthritis treatment, antispasmodic agent, ACE inhibitor, congestion Remover, Antibiotic, Antianginal, Diuretic, Antiparkinsonian, Bronchodilator, Labor Accelerator, Antidiuretic, Antihyperlipidemic, Immunosuppressant, Immunomodulator, Antiemetic, Anti-infective agent, anti-neoplastic agent, anti-fungal agent, anti-viral agent, anti-diabetic agent, anti-allergic agent, antipyretic agent, anti-tumor agent, anti-gout agent, anti-histamine agent, antidiarrheal agent, bone regulator, cardiovascular agent, Cholesterol lowering drugs, antimalarials, quit smoking Drug, antitussive, expectorant, mucolytic agent, nasal congestion agent, dopaminergic agent, gastrointestinal agent, muscle relaxant, neuromuscular blocking agent, parasympathomimetic agent, prostaglandin, stimulant, appetite suppressant , A thyroid agent or an antithyroid agent, a hormone, an anti-migraine agent, an anti-obesity agent, an anti-inflammatory agent and the like.

  In one particularly preferred embodiment, the drug that can be introduced into the cell is a nucleic acid. The nucleic acid is not particularly limited, and may be any DNA, RNA, chimeric nucleic acid of DNA and RNA, 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 contains a nucleotide having a configuration allowing 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 Dithioates, etc., such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, etc.) and sugars (eg, monosaccharides), etc., side-current groups, intercurrent compounds ( For example, acridine, psoralen, etc.), chelation Containing materials (eg, metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, or having modified bonds (eg, α-anomeric nucleic acids) It may be.

  For example, the type of DNA can be appropriately selected according to the purpose of use, and is not particularly limited. Examples thereof include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, and preferably plasmid DNA. , CDNA and antisense DNA. Circular DNA such as plasmid DNA can be appropriately digested with a restriction enzyme or the like and used as linear DNA. The type of RNA can be appropriately selected according to the purpose of use and is not particularly limited. For example, siRNA, miRNA, shRNA, antisense RNA, messenger RNA, single-stranded RNA genome, double-stranded RNA genome , RNA replicon, transfer RNA, ribosomal RNA, and the like, preferably siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon.

The size of the nucleic acid is not particularly limited, and a low molecular nucleic acid (for example, about 5 bp in size) is introduced from a large nucleic acid molecule (for example, about 10 ⁷ kbp in size) such as a chromosome (artificial chromosome, etc.). However, in view of the efficiency of introducing a nucleic acid into a cell, it is preferably 15 kbp or less. For example, the size of a high molecular nucleic acid such as plasmid DNA is 2 to 15 kbp, preferably 2 to 10 kbp. Moreover, as a magnitude | size of a comparatively low molecular nucleic acid like siRNA, 5-1000 bp, Preferably it is 10-500 bp, More preferably, 15-200 bp is illustrated.

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.

A complex of a drug and a cationic molecule (hereinafter sometimes referred to as a cationic complex) that constitutes the drug delivery complex of the present invention is obtained by electrostatic interaction of the cationic complex with an anionic molecule. As long as it can form a drug delivery complex that is substantially uncharged or has a negative surface charge, for example, (a) a drug and a cationic molecule (cationic polymer or A complex having a positive surface charge (hereinafter sometimes referred to as a self-assembled complex) obtained by self-assembly by mixing with liposomes and micelles composed of cationic lipids), (b) a drug Examples include, but are not limited to, cationic micelles (microspheres) encapsulated and (c) cationic liposomes encapsulating drugs. In the case of liposomes, it may be either a monolayer type or a multi-layer type. In addition to the cationic lipid molecules, micelles and liposomes may contain per se known neutral lipids (phospholipids, peptide lipids, glycolipids, etc.), polyoxyalkylene glycols (PEG, PPG, etc.) and the like as constituent molecules. . Those skilled in the art can appropriately select the form of the cationic complex according to the physicochemical properties of the drug to be contained. For example, in the case of a water-soluble drug having a negative charge such as a nucleic acid, a peptide, or a protein, the complex of (a) or (c) can be used, and in the case of a fat-soluble drug such as a lipid or a fat-soluble low molecular weight drug The composite of (b) or (c) above can be used. In the case of a water-soluble drug having a positive charge, the complex (c) can be used.
Further, as long as the cationic complex has a positive surface charge, for example, the cationic complex may be a combination of two or more of the above-described forms. For example, the cationic liposome encapsulating the complex (a) or (b) However, the present invention is not limited to this, and any other combination can be used.

  The compounding ratio of the drug and the cationic molecule used for the preparation of the cationic complex of the present invention is not particularly limited, but for example, the self obtained by mixing the negatively charged drug and the cationic molecule in the cationic complex. In the case of an organized complex, the drug has a negatively charged functional group (for example, a phosphate group in a nucleic acid, a carboxyl group in a protein or a peptide, etc.) and a positively charged functional group of the cationic molecule (for example, The molar ratio to the imino group in PEI, the amino group in polylysine or polyarginine, etc.) is 1: 1 to 1:20, preferably 1: 2 to 1:15, more preferably 1: 2 to 1:10. Can be used.

  In the drug delivery complex of the present invention, the cationic complex is encapsulated in the anionic molecule. Here, “encapsulate” means that it is contained inside, for example, a non-encapsulated form in which the surface of a cationic complex is covered with an anionic molecule bonded by electrostatic interaction. Alternatively, it may take a form in which a cationic complex is encapsulated, such as a liposome. In the former case, as long as the drug delivery complex is substantially uncharged or has a negative surface charge, the cationic complex need not be completely coated with anionic molecules. A drug delivery complex is “substantially uncharged” when it does not cause hemagglutination when contacted with blood and when the drug delivery complex is contacted with cells This means that the positive charge is reduced to some extent when the cell viability is at least 50%. Specifically, the surface charge (ζ potential) of the drug delivery complex is +20 mV or less, preferably +10 mV or less, more preferably +5 mV or less, more preferably 0 mV or less, even more preferably −10 mV or less, particularly preferably −15 mV or less. It is. The lower limit of the surface charge (ζ potential) is not particularly limited, but is, for example, −50 mV or more, preferably −40 mV or more, more preferably −30 mV or more.

  The compounding ratio of the cationic molecule and the anionic molecule used in the preparation of the drug delivery complex of the present invention is such that the drug delivery complex is substantially uncharged or has a negative surface charge. Although there is no particular limitation, for example, a functional group having a positive charge of the cationic molecule (for example, an imino group in PEI, an amino group in polylysine or polyarginine, etc.) and a functional group having a negative charge in the anionic molecule ( For example, the molar ratio of γ-PGA, AGA, CS to sulfonic acid group and carboxyl group) is 5: 1 to 1: 5, preferably 4: 1 to 1: 4, more preferably 3: 1 to 1: 4. It is. However, it can be optimized according to the properties of the cationic molecule and is not limited to this ratio.

In the drug delivery complex of the present invention, a drug and a cationic molecule are contacted at an appropriate mixing ratio to form a cationic complex, and the cationic complex and an anionic molecule are further mixed at an appropriate mixing ratio. It can be prepared by contacting.
For example, a cationic complex utilizing an electrostatic interaction between a water-soluble drug having a negative charge such as a nucleic acid and a water-soluble cationic polymer such as PEI can be obtained by combining the drug and the cationic polymer with water or an appropriate substance. It can be prepared by mixing in buffer and incubating at room temperature for 0.5-300 minutes, preferably 1-180 minutes. The drug concentration in the mixture can be appropriately set in consideration of the type and size (molecular weight) of the drug to be used. However, when the drug is a nucleic acid, it is usually in the range of 0.01 to 1000 ng / μL. When the drug is normal plasmid DNA (size is about 3 kbp), the DNA concentration in the mixed solution is preferably in the range of 50 to 500 ng / μL. If the concentration is too low, the DNA introduced into the cell cannot express the expected function, and if the concentration is too high, the nucleic acid introduction efficiency decreases. When the drug is RNA, the concentration can be appropriately set in consideration of RNA size and the like. However, when the RNA size is about several kbp, the RNA concentration in the mixed solution is usually 3 to 100 ng / It is in the range of μL. The concentration of the nucleic acid is usually in the range of 1 to 500 nM, particularly when the nucleic acid is as small as about 20 to about 200 bp, such as siRNA. The compounding ratio between the drug and the cationic polymer can be appropriately selected from the above range. The incubation time of the mixed solution can be appropriately selected within the above range depending on the type of drug and cationic polymer used. However, if the incubation time is too short, complex formation between the drug and the cationic polymer is not possible. If sufficient and the incubation time is too long, the complex formed may become destabilized, both of which reduce the efficiency of drug delivery to the cell.

  In the case of a cationic micelle encapsulating a drug, for example, a cationic lipid salt and a fat-soluble drug are dissolved in chloroform, and after thoroughly mixing, chloroform is completely removed using an evaporator and a desiccator. In addition, hydrate overnight. After hydration, cationic micelles containing the drug are prepared by sonication. The mixing ratio between the amount of drug used for preparation and the cationic lipid can be appropriately adjusted depending on the physicochemical properties such as fat solubility of the drug. Moreover, it can prepare by not only this method but a well-known method. The amount of drug to be used and the compounding ratio between the drug and the cationic lipid can be appropriately selected from the above ranges.

In the case of a cationic liposome encapsulating a drug, 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, etc. It can be prepared by a method known per se. The amount of drug to be used and the compounding ratio between the drug and the cationic lipid can be appropriately selected from the above ranges.

  By adding an anionic molecule to the obtained cationic complex solution and incubating at room temperature for 0.5 to 300 minutes, preferably 15 to 60 minutes for self-organization, the cationic complex is converted into an anionic molecule. A coated drug delivery complex can be obtained. If the incubation time is too short, the complex formation between the cationic complex and the anionic molecule will be insufficient, and if the incubation time is too long, the formed complex may be destabilized. The drug delivery efficiency is reduced. The compounding ratio of the cationic molecule and the anionic molecule constituting the cationic complex can be appropriately selected from the above range.

The drug delivery complex thus obtained has a surface that is substantially uncharged or has a negative surface charge. Here, “substantially uncharged” is as described above. Specifically, “substantially uncharged” means that the surface charge (ζ potential) is +20 mV or less and −20 mV or more, preferably +10 mV or less and −10 mV or more, more preferably +5 mV or less and −5 mV or more. The “negative surface charge” is 0 mV or less, preferably −5 mV or less, more preferably −10 mV or less, and further preferably −15 mV or less. The lower limit of the surface charge (ζ potential) is not particularly limited, but is, for example, −50 mV or more, preferably −40 mV or more, more preferably −30 mV or more. The ζ potential of the drug delivery complex can be measured using a commercially available ζ potential measuring device.
Since the drug delivery complex of the present invention is substantially uncharged or negatively charged, even if it is administered systemically such as intravenously, it is like a conventional carrier molecule using a cationic polymer such as PEI or a cationic liposome. It does not cause erythrocyte aggregation. Also, non-specific drug delivery to cells other than the target is reduced.

The drug delivery complex of the present invention has an average particle size of 200 nm or less, preferably 150 nm or less, more preferably 100 nm or less. The particle size distribution and average particle size of the drug delivery complex can be calculated from the scattering intensity distribution obtained using, for example, a dynamic light scattering measurement device.
Conventionally, attempts have been made to improve the migration to organs such as the lungs by increasing the particle size and embolizing the reticuloendothelial system at the target site, but with this technique particles are clogged in capillaries throughout the body. There was a risk of causing embolism. On the other hand, the drug delivery complex of the present invention can achieve sufficient retention with a particle size almost equal to that of the cationic complex and can improve the efficiency of drug delivery to a specific organ. Systemic administration is possible.

  The drug delivery complex of the present invention has significantly reduced cytotoxicity compared to cationic complexes using cationic polymers such as PEI that do not contain anionic molecules, thus minimizing undesirable side effects. Can be suppressed.

  The drug delivery complex of the present invention can be used alone or in combination with a pharmacologically acceptable carrier according to a conventional means and used as a pharmaceutical or reagent composition. When the drug delivery complex is formulated as a reagent composition, the complex may be used as is or for example, water or other physiologically acceptable liquid (eg, saline, phosphate buffered saline). (PBS), an aqueous solvent such as a medium used in normal cell culture (for example, RPMI 1640, DMEM, HAM F-12, Eagle medium, etc.), an organic solvent such as ethanol, methanol, DMSO or a mixture of an aqueous solvent and an organic solvent As a sterile solution or suspension. The composition can appropriately contain physiologically acceptable excipients known per se, vehicles, preservatives, stabilizers, binders and the like.

  When the drug delivery complex is formulated as a pharmaceutical composition, the complex is used as it is or a pharmaceutically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder. And can be produced as an oral preparation (eg, tablet, capsule, etc.) or a parenteral preparation (eg, injection, spray, etc.) by mixing in a unit dosage form generally required for the practice of the formulation. .

Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, swelling such as corn starch and gelatin Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used, and appropriate solubilization aids are used. You may use together with an agent, for example, alcohol (example: ethanol), polyalcohol (example: propylene glycol, polyethyleneglycol), a nonionic surfactant (example: polysorbate ^80TM , HCO-50). As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

  The pharmaceutical composition includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants (eg, ascorbic acid, etc.), etc.

The present invention also provides a method for delivering a drug into the cell, which comprises contacting the drug delivery complex of the present invention with the cell.
The type of cell is not particularly limited, and prokaryotic and eukaryotic cells can be used, but eukaryotic cells are preferred. 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.

A more specific description of the step of bringing the drug delivery complex into contact with the cells is as follows, for example.
That is, the cells are suspended in a suitable medium several days prior to contact with the drug delivery complex and cultured under suitable conditions. Upon contact with the drug delivery complex, the cells may or may not be in the proliferative phase. The culture medium at the time of contact may be a serum-containing medium or a serum-free medium, but the serum concentration in the medium is preferably 30% or less, preferably 20% or less. This is because if the medium contains excess protein such as serum, the contact between the drug delivery complex and the cells may be inhibited.

The cell density at the time of contact is not particularly limited and can be appropriately set in consideration of the cell type and the like, but is usually 0.1 × 10 ^{5 to} 5 × 10 ⁵ cells / mL, preferably 0. 1 × 10 ⁵ to 4 × 10 ⁵ cells / mL, more preferably 0.1 × 10 ^{5 to} 3 × 10 ⁵ cells / mL, more preferably 0.2 × 10 ^{5 to} 3 × 10 ⁵ cells / mL, most The range is preferably 0.2 × 10 ⁵ to 2 × 10 ⁵ cells / mL.

  The drug delivery 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 1 to 72 hours, preferably 1 to 60 hours, more preferably 1 to 48 hours, and still more preferably. The range is 1 to 40 hours, most preferably 1 to 32 hours.
If the culture time is too short, the drug is not sufficiently introduced into the cells, and if the culture time is too long, the cells may be weakened.

Although the drug is introduced into the cells by the above culture, the medium is preferably replaced with a fresh medium, or the culture is further continued by adding a fresh medium to the medium. 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 the function expected of the drug and the like. However, when the drug is a plasmid DNA such as an expression vector, it is usually 8 to 72 hours, preferably Is in the range of 8 to 60 hours, more preferably 8 to 48 hours, still more preferably 8 to 36 hours, and most preferably 12 to 32 hours. When the compound is a low-molecular nucleic acid capable of controlling the expression of a target gene such as siRNA, it is usually 0 to 72 hours, preferably 0 to 60 hours, more preferably 0 to 48 hours, and still more preferably 0 to 36. Time, most preferably in the range of 0 to 32 hours.

The present invention also provides a method for delivering a drug into cells of the animal, which comprises administering the drug delivery complex of the present invention to a subject.
That is, by administering the complex to a subject, the complex reaches / contacts the target cell, and the 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.

  The administration method of the drug delivery complex is not particularly limited as long as the complex reaches and contacts the target cell and the drug contained in the complex can be introduced into the cell. Considering the type and site, etc., per se known administration methods (oral administration, parenteral administration (intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.), etc.) It can be selected appropriately.

  The dose of the drug delivery complex is not particularly limited as long as introduction of the drug into the cell can be achieved, taking into consideration the type of administration target, the administration method, the type of drug, the type and site of the target cell, etc. In the case of oral administration, for example, in the case of a human (with a body weight of 60 kg), the single dose is generally about 0.001 mg to 10,000 mg as a complex. When administered parenterally (for example, intravenous administration), generally, for example, in humans (with a body weight of 60 kg), the single dose is about 0.0001 mg to 3000 mg as a complex.

  After the systemic administration such as intravenous administration, the drug delivery complex of the present invention is γ-PGA, CS or AGA or a salt thereof, preferably γ-PGA or CS or a salt thereof, more preferably γ-PGA or a salt thereof as an anionic molecule. When the salt is used, it is delivered with high selectivity to the spleen.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

Example 1
(Method)
As a plasmid DNA (hereinafter abbreviated as pDNA), pCVM-Luc containing a CMV promoter and encoding a firefly luciferase gene was used. After mixing 10 μL of 5% glucose solution of pDNA (1 mg / mL) and 20 μL of 5% sugar solution, it was mixed with 10 μL of 5% glucose solution (1 mg / mL, pH = 7.4) of PEI (molecular weight 25000) ( The molar ratio of phosphate group of pDNA to amino group of PEI was 1: 8) and incubated at room temperature for 15 minutes. 5% glucose solutions of various concentrations of γ-PGA (molecular weight: 10,000 to 100,000) were added to this mixed solution, and the phosphate group (P) of pDNA, the amino group (N) of PEI and the carboxyl group of γ-PGA ( C) molar ratios (hereinafter abbreviated as “PNC ratio”) were 1: 8: 2, 1: 8: 4, and 1: 8: 6, respectively, and incubated at room temperature for 15 minutes. For each PNC ratio, the surface charge (ζ potential) and particle diameter of the obtained pDNA / PEI / γ-PGA complex were measured using Zetasizer Nano (Nano-ZS, manufactured by Malvern). In addition, chondroitin sulfate and alginic acid were examined by the same method.
(result)
The measurement results of the particle diameter and surface charge are shown in FIG. The bar graph represents the particle diameter, and the line graph represents the surface charge value. In all three types of anionic molecules, those having a PNC ratio of 1: 8: 4 and 1: 8: 6 had a particle size almost equal to that of the pDNA / PEI complex and had a negative surface charge. . Since the surface charge shows a stable anionic property, a complex having a PNC ratio of 1: 8: 6 was used in subsequent gene introduction experiments unless otherwise stated.

Example 2
(Method)
The three pDNA / PEI / γ-PGA complexes prepared in Example 1 and the pDNA / PEI / CS (molecular weight of 100,000 or less) complex prepared in the same manner as in Example 1 (PNC ratio (where C is This means the number of moles of carboxyl groups and sulfonic acid groups in CS (also in the following examples) 1: 8: 6) and pDNA / PEI / AGA (molecular weight 12000 to 80000) complex (PNC ratio 1: 8). : 6) was mixed with erythrocyte solution (2%, v / v) and incubated at room temperature for 15 minutes to examine whether or not aggregation was induced. The measurement was performed under a microscope (400 times).
(result)
The results are shown in FIG. When contacted with the pDNA / PEI complex, erythrocytes aggregated, but the pDNA / PEI / γ-PGA complex, pDNA / PEI / CS complex, and pDNA / PEI / AGA complex all induce erythrocyte aggregation. I did not.

Example 3
(Method)
4 types of molecular weight (A type: 1,500,000, B type: 500,000 to 800,000, C type: 200,000 to 400,000, D type: 10,000 to 100,000) Using PGA, pDNA / PEI / γ-PGA complexes (PNC ratios 1: 8: 2, 1: 8: 4, 1: 8: 6, respectively) were prepared in the same manner as in Example 1. Each complex was subjected to 0.8% agarose gel electrophoresis, and the gel was stained with EtBr after the electrophoresis.
(result)
The results are shown in FIG. When A-type, B-type, and C-type γ-PGA was used, pDNA efflux was observed in the PNC ratio 1: 8: 4 and 1: 8: 6 complexes, but D-type γ-PGA was In the complex used, no pDNA efflux was observed at all PNC ratios examined. D-type γ-PGA was used in the subsequent experiments unless otherwise specified, because it showed excellent pDNA retention ability regardless of the mixing ratio.

Example 4
(Method)
Using the D type γ-PGA of Example 3 and α-PGA having a molecular weight approximate to that of the D type, the pDNA / PEI / γ- or α-PGA complex (respectively PNC The ratios were 1: 8: 2, 1: 8: 4, 1: 8: 6, 1: 8: 8), and the pDNA retention ability of the complex was examined in the same manner as in Example 3. Similarly, pDNA / PEI / CS or AGA complexes (PNC ratios of 1: 8: 2, 1: 8: 4, 1: 8: 6, respectively) were also prepared and examined for pDNA retention ability.
(result)
The results are shown in FIG. When α-PGA was used, pDNA efflux was observed as the blending ratio of α-PGA was increased, but when γ-PGA was used, pDNA efflux was not observed at all PNC ratios examined. . Even when CS or AGA was used as the anionic molecule, no pDNA efflux was observed in all PNC ratios examined.

Example 5
(Method)
Using pEGFP-C1 encoding green fluorescent protein (GFP) as pDNA, PEI (R-PEI) fluorescently labeled with rhodamine B, and various polyanions (poly A, poly IC, fucoidan, λ-carrageenan, xanthan, α -A pDNA / R-PEI / polyanion complex was prepared in the same manner as in Example 1 using polyaspartic acid, α-PGA, γ-PGA, CS, AGA). Each complex was brought into contact with B16 / F10 melanoma cells (source: Tohoku University Institute of Aging Medicine) for 2 hours in the absence of serum, and after culturing for another 22 hours after contact, fluorescence under a fluorescence microscope was observed. .
(result)
The results are shown in FIGS. The upper row shows the color development due to cellular uptake, and the lower row shows the color development due to gene expression. The pDNA / R-PEI complex, the pDNA / R-PEI / γ-PGA complex, the pDNA / PEI / CS complex, and the pDNA / PEI / AGA complex were observed to develop color in both upper and lower stages, but other complexes In the upper and lower stages, no color development was observed.

Example 6
(Method)
A pDNA / PEI / polyanion complex was prepared in the same manner as in Example 5 except that pCMV-Luc encoding a firefly luciferase gene was used as pDNA, and non-labeled PEI was used, and the pDNA / PEI / polyanion complex was transferred to B16 / F10 melanoma cells. And gene expression was quantified. Further, cytotoxicity was evaluated by WST-1 assay (Reference: In Vitro Toxicol. 8, 187-190 (1995)).
(result)
The degree of gene expression is shown in FIG. 7, and the degree of cytotoxicity is shown in FIG. From FIG. 7, the luciferase expression by the complex using γ-PGA or CS as the anionic molecule was sufficiently low compared with the pDNA / PEI complex, but at a sufficiently high level. Even when AGA was used, the expression level was lower than that of γ-PGA and CS, but an expression level sufficient for use as a gene transfer reagent was observed. Further, from FIG. 8, the pDNA / PEI / γ-PGA, CS or AGA complex has a significantly higher cell viability than the pDNA / PEI complex, and these anionic molecules are used for the pDNA / PEI complex. It has been shown that the cytotoxicity is greatly improved by coating.

Example 7
(Method)
As described in Example 6, pDNA / PEI / polyanion complexes were prepared so that the PNC ratios were 1: 8: 4, 1: 8: 6, and 1: 8: 8 for γ-PGA, CS, and AGA, respectively. Then, luciferase expression when transfected into B16 / F10 melanoma cells was measured and compared.
(result)
The results are shown in FIG. In any anionic molecule, almost the same gene expression was observed within the range of the PNC ratio examined.

Example 8
(Method)
40 μg of pDNA / PEI complex, pDNA / PEI / γ-PGA complex and pDNA / PEI / CS complex were administered as a pDNA amount in 3 ddY male mice (5 weeks old, obtained from SLC) as the amount of pDNA. 6, 12 and 24 hours later, the liver, kidney, spleen, heart and lung were removed and luciferase expression in each organ was measured.
(result)
The results are shown in FIG. When pDNA / PEI / γ-PGA or CS complex was administered, luciferase expression in the lung was remarkably suppressed as compared with pDNA / PEI complex, and only a high level exceeding 10 ⁶ RLU per g organ was observed only in the spleen. Expression was observed, and it was shown that gene transfer into the spleen can be performed with high selectivity even by systemic administration by using γ-PGA or CS.

Example 9
(Method)
A pDNA / cationic molecule / γ-PGA complex (PNC ratios 1: 8: 2, 1: for each) in the same manner as in Example 1 except that polylysine, polyarginine, and protamine are used instead of PEI as the cationic molecule. 8: 4, 1: 8: 6), and the pDNA retention ability was assayed in the same manner as in Example 3.
(result)
The results are shown in FIG. No pDNA efflux was observed in all the complexes, and it was shown that pDNA was stably encapsulated by coating with γ-PGA regardless of the type of cationic molecule.

Example 10
(Method)
Using pCMV-Luc encoding firefly luciferase gene as pDNA, using polylysine, polyarginine or protamine as the cationic molecule and γ-PGA as the anionic molecule, a pDNA / cationic molecule / γ-PGA complex was prepared. In the same manner as in Example 6, B16 / F10 melanoma cells were transfected, and gene expression was quantified.
(result)
The results are shown in FIG. Whichever cationic molecule was used, the complex coated with γ-PGA showed the same expression as the uncoated cationic complex.

Example 11
(Method)
The pDNA was made into typical cationic liposomes, DOTMA-DOPE or DOTMA-CHOL, mixed with the liposomes, and incubated at room temperature for 15 minutes to form a complex. Furthermore, this pDNA-encapsulated liposome suspension was mixed with a γ-PGA solution and incubated at room temperature for 15 minutes to prepare a complex in which the liposome was coated with γ-PGA (PNC ratio 1: for each). 2: 2-12, where N means the number of moles of trimethylammonium groups in DOTMA). These complexes were electrophoresed in the same manner as in Example 3. In addition, luciferase activity was measured by bringing these complexes into contact with cells in the same manner as in Example 6.
(result)
The result of electrophoresis is shown in FIG. 13, and the measurement result of luciferase activity is shown in FIG. As shown in FIG. 13, in any case of using any of the cationic liposomes, pDNA outflow from the complex was not observed in all PNC ratio ranges examined, and it was found that pDNA was stably encapsulated. It was. Further, from FIG. 14, it was clarified that any cationic liposome coated with γ-PGA showed the same gene expression as the uncoated liposome.

Example 12
(Method)
The DOTMA-CHOL liposome or 1-amide decane micelle prepared by a conventional method was prepared, and the γ-PGA solution was mixed at various ratios, and incubated at room temperature for 30 minutes. A coated composite was made. These complexes were electrophoresed in the same manner as in Example 3.
(result)
The results are shown in FIG. In the figure, the top is the minus pole and the bottom is the plus pole. Both the cationic liposome and the cationic micelle migrate to the negative electrode side as they are, but it was confirmed that they were transferred to the positive electrode side by coating with γ-PGA. Therefore, it became clear that γ-PGA can encapsulate both cationic liposomes and cationic micelles.

  Since the drug delivery complex of the present invention does not cause hemagglutination, has low cytotoxicity, and is excellent in intracellular uptake efficiency, it is useful as a vector for safely and effectively delivering a drug to a cell to a target cell. Further, since selective drug delivery to the spleen is possible even by systemic administration such as intravenous administration, it is useful as a simple drug targeting means for the spleen.

It is a figure which shows the measurement result of the particle diameter and surface charge in Example 1. FIG. The bar graph shows the particle diameter, and the line graph shows the surface charge. It is a figure which shows the mode of erythrocyte aggregation in Example 2. FIG. FIG. 6 is a diagram showing the result of electrophoresis in Example 3. It is a figure which shows the result of the electrophoresis in Example 4. It is a figure which shows the mode of intracellular uptake | capture of pDNA in Example 5, and the mode of gene expression. The upper row shows cellular uptake, and the lower row shows color development due to gene expression. It is a figure which shows the mode of intracellular uptake | capture of pDNA in Example 5, and the mode of gene expression. The upper row shows cellular uptake, and the lower row shows color development due to gene expression. It is a figure which shows the luciferase activity in Example 6. 10 is a graph showing the degree of cytotoxicity in Example 6. FIG. It is a figure which shows the luciferase activity in Example 7. It is a figure which shows the result of having measured the luciferase activity in a liver, a kidney, a spleen, a heart, and a lung after 6 hours, 12 hours, and 24 hours in Example 8. The vertical axis is the numerical value of luciferase activity. 10 is a diagram showing the results of electrophoresis in Example 9. FIG. It is a figure which shows the luciferase activity in Example 10. 10 is a diagram showing the results of electrophoresis in Example 11. FIG. It is a figure which shows the measurement result of the luciferase activity in Example 11. It is a figure which shows the result of the electrophoresis in Example 12. FIG.

Claims (9)

A drug delivery complex comprising a complex of a drug and a cationic molecule and an anionic molecule encapsulating the complex and having a substantially uncharged or negative surface charge, wherein the anionic molecule is γ− poly-glutamic acid, Ru salts thereof der you and, drug delivery complex. The complex of a drug and a cationic molecule is selected from the group consisting of a complex by self-assembly of a drug and a cationic molecule, a cationic micelle encapsulating the drug, and a cationic liposome encapsulating the drug. 1 Symbol placement drug delivery complex of. The drug delivery complex according to claim 1 or 2 , wherein the drug is selected from the group consisting of nucleic acids, peptides, proteins, polysaccharides and low molecular weight compounds. The drug delivery according to any one of claims 1 to 3 , wherein the molar ratio of the functional group having a positive charge of the cationic molecule and the functional group having a negative charge of the anionic molecule is 3: 1 to 1: 4. Complex. The drug delivery complex according to any one of claims 1 to 4 , wherein the molecular weight of the anionic molecule is 100,000 or less. The drug delivery complex according to any one of claims 1 to 5 , which is for delivering a drug to the spleen. The drug delivery complex according to any one of claims 1 to 6 , which can be administered systemically. A method for delivering a drug into a cell, comprising contacting the drug delivery complex according to any one of claims 1 to 5 with a cell other than in vitro or a human individual. A method for delivering a drug into a cell of an animal, comprising administering the drug delivery complex according to any one of claims 1 to 7 in vitro or to a mammal other than a human.