WO2011052804A1

WO2011052804A1 - Method of delivering agent into target cell

Info

Publication number: WO2011052804A1
Application number: PCT/JP2010/069767
Authority: WO
Inventors: Makoto Haga; Naruyoshi Fujiwara; Akihiro Nishi; Satoru Kubo; Takanori Kushibiki
Original assignee: Tokyo University Of Science Educational Foundation Administrative Organization
Priority date: 2009-10-30
Filing date: 2010-10-29
Publication date: 2011-05-05

Abstract

The present invention relates to a method of delivering an agent into a target cell, including contacting an agent-encapsulating liposome with the target cell in a serum-free isotonic solution that has a pH of from 5 to 9 and contains Fe³⁺ or La³⁺ ions at a concentration of from 100 μM to 3000 μM, so as to deliver the agent into the target cell.

Description

DESCRIPTION

METHOD OF DELIVERING AGENT INTO TARGET CELL

Technical Field

[0001] The present invention relates to a method of delivering an agent into a target cell. Background Art

[0002] Liposomes are composed of biodegradable lipids that derive from living organisms, and application thereof to drug delivery systems (DDSs) has thus been proposed. In a case in which liposomes are applied to DDSs, it is a target to improve the efficiency of the incorporation of the agents encapsulated in liposomes into cells.

[0003] In order to achieve the target, measures have been taken such as optimization of the particle diameters of the liposomes (see, for example, Eur. J Pharm. Biophrm.(2008)

71:409-19 and J. Liposome. Res. (2007) 17:197-203).

[0004] Further, liposomes having several types of specific structure that have been reported to be capable of being efficiently uptaken by cells by endocytosis are known (see, for example, Bioconjugate. Chem. (2008) 19:1588-1595 and J. Control. Rel. (2008) 130:77-83). However, these liposomes are transferrin-binding liposomes, which are liposomes having relatively large particle diameters and devised so as to facilitate endocytosis. Therefore, it is difficult to apply these liposomes to DDSs of which the object is to transfer agent-encapsulating liposomes into target tissues by exuding from blood vessels.

[0005] With regard to the properties of liposomes, it has been reported that addition of trivalent lanthanum (La) causes membrane fusion between liposomes with high frequency, (see, for example, Biochim. Biophys. Acta. (2001) 1515:189-201 and Langmuir (2004) 20:5160-5164).

However, sufficient study has not been conducted with respect to methods of efficiently delivering agents encapsulated in liposomes into cells.

SUMMARY OF INVENTION

Technical Problem

[0006] An object of the present invention is provision of a method of efficiently delivering an agent encapsulated in a liposome into a target cell.

Solution to Problem

[0007] Aspects of the present invention include the following. <1> A method of delivering an agent into a target cell, comprising contacting an agent-encapsulating liposome with the target cell in a serum-free isotonic solution that has a pH of from 5 to 9 and contains Fe³⁺ or La³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ, so as to deliver the agent into the target cell.

<2> The method of delivering an agent into a target cell according to <1>, wherein the serum-free isotonic solution is a phosphate buffer solution that has a pH of from 5 to 9 and contains Fe³⁺ or La³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ.

<3> The method of delivering an agent into a target cell according to <1>, wherein the serum-free isotonic solution is a Good's buffer that has a pH of from 5 to 9 and contains Fe³⁺ or La³⁺ ions at a concentration of from 500 μΜ to 3000 μΜ.

[0008] <4> The method of delivering an agent into a target cell according to <3>, wherein the Good's buffer is a HEPES solution.

<5> The method of delivering an agent into a target cell according to any one of <1> to <4>, wherein the serum-free isotonic solution is a serum-free isotonic solution that has a pH of from 5 to 9 and contains Fe³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ.

<6> The method of delivering an agent into a target cell according to any one of <1> to <5>, wherein the agent is of at least one type selected from the group consisting of a nucleic acid, a drug, a chemical substance and a protein.

[0009] <7> The method of delivering an agent into a target cell according to <6>, wherein the drug is an anticancer agent.

<8> The method of delivering an agent into a target cell according to <6>, wherein the chemical substance is a differentiation inducing agent or a reprogramming agent.

<9> The method of delivering an agent into a target cell according to any one of <1> to <8>, wherein the target cell is of at least one type selected from the group consisting of a cancer cell, a tissue-derived cell, an organ-derived cell, and a stem cell.

[0010] <10> The method of delivering an agent into a target cell according to any one of <1> to <9>, wherein the target cell is a cancer cell, and the agent is an anticancer agent.

<11> The method of delivering an agent into a target cell according to any one of <1> to <9>, wherein the target cell is a stem cell, and the agent is a differentiation inducing agent.

<12> The method of delivering an agent into a target cell according to any one of <1> to <9>, wherein the target cell is a tissue-derived cell or an organ-derived cell, and the agent is a reprogramming agent.

[0011] <13> A serum-free isotonic solution containing Fe ions at a concentration of from 100 μΜ to 3000 μΜ and having a pH of from 5 to 9. <14> The serum-free isotonic solution according to <13>, wherein the serum-free isotonic solution is a phosphate buffer solution containing Fe³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ and having a pH of from 5 to 9.

<15> The serum-free isotonic solution according to <13>, wherein the serum-free isotonic solution is a Good's buffer containing Fe³⁺ ions at a concentration of from 500 μΜ to 3000 μΜ and having a pH of from 5 to 9.

[0012] <16> The serum-free isotonic solution according to <15>, wherein the Good's buffer is a HEPES solution.

<17> The serum-free isotonic solution according to any one of <13> to <16>, wherein the serum-free isotonic solution is used for delivering an agent into a target cell.

<18> A pharmaceutical composition comprising the serum-free isotonic solution of <13> and an agent-encapsulating liposome.

[0013] <19> An agent-delivering kit comprising a section including the serum-free isotonic solution of <13> and a section including an agent-encapsulating liposome.

BRIEF DESCRIPTION OF DRAWINGS

[0014] Fig. 1 shows an effect of Fe(3+) ion concentration on the uptake of liposomes by 4T1 cells. Data represent mean ±S.E. (n=3). *p<0.05, **p<0.01 compared with control by paired t-test.

[0015] Fig. 2 shows an effect of different inhibitors on the uptake of liposomes by 4T1 cells. Cells were pre-incubated with inhibitors for 30 min at 37°C. Concentration of Fe(3+) ions was 1 mmol/L. The fluorescence intensity of cells in the absence of inhibitor was set to 100%. Data represent mean ±S.E. (n=3).

[0016] Fig. 3 shows uptake of rhodamin-PE-labeled liposomes encapsulating calcein by 4T1 cells 60 min after the start of incubation in the absence (upper images) and presence (lower images) of Fe(3 +) ions.

[0017] Fig. 4 shows uptake of liposomes by various cell lines induced by Fe(3+) ions.

Data represent mean ±S.E. (n=3). *p<0.05, **p<0.01 compared with control by paired t-test.

[0018] Fig. 5 shows an effect of Fe(3+) ion concentration on GFP expression and cell viability. Data represent mean ±S.E. (n=3). *p<0.05, **p<0.01 compared with control by paired t-test.

[0019] Fig. 6 shows comparison of GFP expression between Fe(3+) ions and DOTAP reagent. Data represent mean ±S.E. (n=3). *p<0.05, **p<0.01 compared with control by paired t-test. [0020] Fig. 7 shows uptake of calcein-encapsulating liposomes by 4T1 cells in the presence of Fe(3+) or La(3+) ions. Histogram in FACS analysis.

[0021] Fig. 8 shows uptake of calcein-encapsulating liposomes by suspended 4T1 cells in the presence of FBS (a) and in the absence of FBS (b). Data represent mean ±S.E. (n=3). Student's test *p<0.05, **p<0.01 vs. control.

[0022] Fig. 9 shows uptake of calcein-encapsulating liposomes by adherent 4T1 cells in the presence of FBS (a) and in the absence of FBS (b) Data represent mean ±S.E. (n=3).

Student's test *p<0.05, **p<0.01 vs. control.

[0023] Fig.10 shows uptake of calcein-encapsulating SUV by 4T1 cells during 60 min at 37°C. (a) Histogram in FACS analysis, (b) Uptake efficiency.

[0024] Fig.11 shows effects of Fe (3+) (final concentration: lmmol/L) and Lipofectamine 2000 on GFP expression in 4T1 cells, (a) Histgram of FACS analysis (b) GFP expression.

[0025] Fig.12 shows effects of Fe (3+) (final concentration: 2mM) and Lipofectamine 2000 on GFP expression in COS-7 cells, (a) Histogram of FACS analysis (b) GFP expression.

[0026] Fig.13 shows an effect of pH in PBS solution on the GFP expression in 4T1 cells. Control experiment was performed in 300mmol/L sucrose aqueous solution containing 5mmol/L HEPES (pH7.4)

[0027] Fig.14 shows an effect of buffer solution containing Fe (3+) on the GFP expression in 4T1 cells. Control experiment was performed in 300mmol/L sucrose aqueous solution containing 5mmol/L HEPES (pH7.4)

[0028] Fig.15 shows GFP expression in 4T cells in PBS or HEPES solution at low Fe (3+) concentration.

[0029] Fig.16 shows dependence of GFP expression in 4T1 cells on Fe (3+) concentration in HEPES solution.

DESCRIPTION OF EMBODIMENTS

[0030] An aspect of the present invention provides a method of delivering an agent into a target cell, the method including contacting an agent-encapsulating liposome with the target cell in a serum-free isotonic solution that has a pH of from 5 to 9 and contains Fe³⁺ or La³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ, so as to deliver the agent into the target cell.

[0031] According to the invention, the agent can be efficiently delivered into the target cell by adjusting the concentration of Fe³⁺ ions or La³⁺ ions to be in the range of from 100 μΜ to 3000 μΜ.

The present invention is described in detail below. Although the below descriptions of the constituent elements sometimes refer to representative embodiments of the invention, the invention is by no means limited to the embodiments.

[0032] <Agent-encapsulating Liposome>

In the invention, lipids for forming liposomes are not particularly limited, and examples thereof include: phospholipides such as natural phosphatidylcholine, synthetic phosphatidylcholine, natural phosphatidylethanolamine, synthetic phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and phosphatidic acid; and glycolipids such as sphingoglycolipids and glyceroglycolipids. Examples of the natural phosphatidylcholine include egg yolk phosphatidylcholine (EPC, L-a-phosphatidylcholine) and soy bean phosphatidylcholine, and examples of the synthetic phosphatidylcholine include dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylcholine (DOPC). Examples of the naturalphosphatidylethanolamine include egg yolk phosphatidylethanolamine, and examples of the synthetic phosphatidylethanolamine include

dipalmitoylphosphatidylethanolamine. Examples of the phosphatidylglycerol include dipalmitoylphosphatidylglycerol. Phospholipids are preferable, and egg yolk

phosphatidylcholine (EPC) is particularly preferable.

[0033] These lipids may be used singly, or in combination of two or more thereof. At least one of these lipids may be used in combination with at least one non-polar substance such as a cholesterol. For example, a mixed lipid containing egg yolk phosphatidylcholine (EPC) and cholesterol (Choi) may be used. The materials for forming liposomes preferably include at least one of dicetyl phosphate (DCP), which is a charged substance, a-tocopherol, which is an antioxidant, or the like.

[0034] In a case in which one or more phospholipids and one or more cholesterols are used in combination, the molar ratio of the phospholipids to the cholesterols (phospholipids:

cholesterols) is preferably in the range of approximately from 2:1 to 1:1. However, the ratio is not limited thereto.

The form of the liposome in the invention is not particularly limited, and may be any of a multilamellar liposome (MLV), small unilamellar liposome (SUV), or a large unilamellar liposome (LUV).

[0035] In the invention, the average particle diameter of liposomes is preferably from 80 run to 300 nm, more preferably from 90 nm to 210 nm, and still more preferably from 100 run to 200 nm.

The method of measuring the particle diameters of liposomes may be, for example, a dynamic light scattering method (see Liposome Technology, 2nd Edition Vol.1 Chapter 15 p254-269 (1993); CRC Press, Edited by Gregory Gregoriadis). In a case in which the particle diameters of the liposomes are significantly uneven, the particle diameters are preferably measured by observation with negative staining using a transmission electron microscope (Biochimica et Biophysica Acta, 601 (1980) 559-571), cryo-electron microscopy imaging (Liposome Technology, 2nd Edition Vol.2 Chapter 14 p250-252 (1993); CRC Press, Edited by Gregory Gregoriadis), or an atomic force microscope (Thin solid films 273 (1996) 297-303).

[0036] The method of producing the liposome in the invention may be any known production method as long as the object of the invention can be achieved. Examples of the method include surfactant removal method, hydration method, ultrasonic method,

reversed-phase distillation method, freezing-melting method, ethanol-injection method, extrusion method, and high-pressure emulsification method (see, for example, Japanese Patent Application Laid-Open (JP-A) No. 9-208599 and Biochim. Biophys. Res. Commun., 224, 242-245,1996).

[0037] The liposome in the invention is a liposome that encapsulates an agent.

The agent encapsulated in the liposome is not particularly limited. The liposome may encapsulate an agent that is desired to be delivered into the target cell. Examples of the agent to be encapsulated in the liposome include a nucleic acid, a drug, a chemical substance or a protein. Examples of the gene includes a single strand or double strand DNA, a cDNA, a genomic DNA, a single strand or double strand RNA. The mixing weight ratio of the encapsulated agent and the liposomal lipid is preferably from 1 : 100 to 1:50. When the encapsulated agent is a protein, the molecular weight of the protein is preferably from 1000 Da to 100,000 Da, and more preferably from 1000 Da to 80,000 Da.

Examples of the agent (the desired agent) to be encapsulated in the liposome also include a differentiation inducing agent and a reprogramming agent. Specific examples of the reprogramming substance include Oct4, Sox2, Klf4 and c-Myc.

[0038] Examples of the drug include nucleic acids,_. polynucleotides, genes, and analogues thereof; glycosaminoglycans and derivatives thereof; oligosaccharides, polysaccharides, and derivatives thereof; proteins and peptides; an anti-inflammatory agent, a steroid

anti-inflammatory agent, an anticancer agent, an enzyme preparation, an enzyme inhibitor, an antibiotic, an antioxidant, a lipid incorporation inhibitor, a hormone preparation, an angiotensin-converging enzyme inhibitor, an angiotensin receptor agonist, a smooth muscle cell proliferation/migration inhibitor, an antiplatelet drug, a chemical mediator release inhibitor, a vascular endothelial cell proliferation inducing agent, a vascular endothelial cell proliferation inhibitor, an aldose reductase inhibitor, a mesangial cell proliferation inhibitor, a lipoxygenase inhibitor, an immunosuppressive drug, an adjuvant, an antiviral agent, an anticoagulant agent, a vasodilator agent, a Maillard reaction inhibitor, an amyloidosis inhibitor, an NOS inhibitor, an AGEs inhibitor and an radical scavenger; and an in vivo diagnostic agent.

[0039] In the invention, it is not essential that the liposome encapsulates only one agent, and plural agents may be encapsulated in the same liposome. The scope of the invention also encompasses an embodiment in which plural types of liposome, each of which encapsulates a different agent, are simultaneously present in the serum-free isotonic solution.

The method of encapsulating the agent in the liposome is not particularly limited, and methods available to a person in the art area all applicable. In the invention, plural agents may be incorporated into inside the liposome in portions by conducting incorporation operation plural times.

[0040] For example, the agent may be encapsulated in the liposome by adding the agent as an aqueous solution at the time of forming the liposome. It is also possible to employ a method whereby a concentration gradient such as a pH gradient is formed between the outside the vesicle and the inside of the vesicle after the formation of the liposome (vesicle), and an ionizable agent is incorporated into inside the liposome due to the resultant potential as a driving force (see Cancer Res., 49, 5922, 1989 and BBA, 455, 269, 1976).

[0041] <Target Cell>

The target cell in the invention is not particularly limited, and may be a cell collected from a biological subject or a cultured cell line. Examples of the target cell include a cell from a cancer, tissue or organ. Examples of the cell line include 4T1, HeLa, HEK293, B16BL6, RAW246.7 and COS-7. The target cell may be a stem cell such as an embryonic stem (ES) cell, an embryonal carcinoma (EC) cell, an embryonic germ (EG) cell, an induced pluripotent stem (iPS) cell, or an adult stem cell such as a tissue stem (TS) cell or a somatic stem cell.

The target cell in the invention is preferably a cancer cell, a tissue-derived cell, an organ-derived cell, or a stem cell.

The scope of the invention encompasses an embodiment in which one type of target cell is present in the serum-free isotonic solution, as well as an embodiment in which various types of target cell, such as those described above, are present as a mixture in the serum-free isotonic solution.

[0042] <Serum-free Isotonic Solution>

In the invention, the pH of the serum-free isotonic solution containing a Fe ion or a La³⁺ ion is from pH5 to pH9, and more preferably from pH5.5 to pH8.5. A pH in the range of from pH5 to pH9 allows the activity of the target cell in the serum-free isotonic solution to be maintained, and also allows the agent to be efficiently delivered into the target cell. A preferable range of the pH of the serum-free isotonic solution in the invention varies with the type of the target cell to be used, and may be selected as appropriate within the range of from pH5 to pH9.

Examples of the serum-free isotonic solution in the invention include a phosphate buffer solutions (e.g., PBS) and Good's buffers. Among Good's buffers, solutions classified as N-substituted taurines are preferable, and HEPES solution is particularly preferable.

[0043] In a case in which a phosphate buffer solution is used as the serum-free isotonic solution, the concentration of Fe ions or La ions is preferably from 300 μΜ to 3000 μΜ, and more preferably from 800 μΜ to 2000 μΜ.

When a Good's buffer such as HEPES solution is used as the serum-free isotonic solution in the invention, the concentration of Fe ions or La ions is preferably from 500 μΜ to 3000 μΜ, and more preferably from 1 mM to 2 mM.

[0044] The serum-free isotonic solution in the invention may be a solution that can maintain cell growth and perfect nutrition without addition of serum, as long as the effect with respect to efficient delivery of the agent into the target cell is not affected. However, the serum-free medium may contain refined nature or recombination protein, a growth factor, etc.

Examples of the serum-free medium include DMEM, RPMI 1640, IMDM, MEM, EBSS, etc.

The serum-free isotonic solution in the invention may contain salts, amino acid, sugar, vitamin, etc. as long as the effect with respect to efficient delivery of the agent into the target cell is not affected.

[0045] <Fe³⁺ Ion or La³⁺ Ion>

The concentration of Fe ions or La ions in the serum-free isotonic solution in the invention is from 100 μΜ to 3000 μΜ. A concentration of Fe ions or La³⁺ ions within the above range allows the agent to be delivered into the target cell efficiently.

In a case in which a phosphate buffer solution is used as the serum-free isotonic solution, the concentration of Fe ions or La ions is preferably from 300 μΜ to 3000 μΜ, and more preferably from 800 μΜ to 2000 μΜ. In a case in which a HEPES solution is used as the serum-free isotonic solution, the concentration of Fe ions or La³⁺ ions is preferably from 500 μΜ to 3000 μΜ, and more preferably from ImM to 2mM.

It is most preferable to use Fe³⁺ ions from the viewpoint of efficiently delivering the agent into the target cell.

[0046] <Method of Delivering Agent into Target Cell>

The method of delivering the agent into the target cell in the invention is not particularly limited as long as the incorporation rate of the agent encapsulated in the liposome into the target cell is high as compared to conventionally-employed agent-delivery systems such as DOTAP Liposomal Transfection Regent (tradename, manufactured by Roche Applied Science ) and LIPOFECTAMINE (tradename, manufactured by Invitrogen Corporation).

In the invention, the contact between the agent-encapsulating liposome and the target cell is not particularly limited in terms of the manner thereof, as long as the contact results in incorporation of the agent encapsulated in the liposome into the target cell. Examples thereof include fusion of the agent-encapsulating liposome membrane and the cell membrane of the target cell, and incorporation of the agent-encapsulating liposome into the target cell by endocytosis by the target cell.

[0047] In the invention, contacting the agent-encapsulating liposome and the target cell may be, for example, allowing the agent-encapsulating liposome and the target cell to be present in the same serum-free isotonic solution.

The manner of delivering the agent into the target cell in the invention is not particularly limited as long as the presence of the agent originally encapsulated in the liposome becomes to be found in the target cell.

In the invention, the agent may be delivered, in portions, into the target cell plural times. The scope of the invention also encompasses an embodiment in which different agents are simultaneously delivered into the target cell.

[0048] Pharmaceutical Composition>

The agent-encapsulating liposome in the invention may be used for the treatment of various diseases such as cancer, brain infarction, arteriosclerosis, and sepsis.

Specifically, the agent-encapsulating liposome can be administered to a patient in the form of a pharmaceutical composition or formulation obtained by mixing with a

pharmaceutically acceptable carrier (such as an emulsifier, a diluent, or a solubilization agent), using a method such as intravascular administration, intravesical (bladder) administration, intraperitoneal administration, or topical administration.

[0049] The agent-encapsulating liposome in the invention can be formulated into a pharmaceutical formulation according to a known method such as the methods described in European Patent Application Publication Nos. 526700 and 520499.

With regard to the dose of the agent-encapsulating liposome in the invention, an optimal dose can be determined based on the type of the agent encapsulated in the liposome. For example, in a case in which adriamycin is encapsulated as the agent, the dose in terms of the adriamycin amount may be 50 mg/kg or less, preferably 10 mg/kg or less, and more preferably 5 mg/kg or less. [0050] <Kit>

The configuration of the kit for incorporating an agent into a target cell in the invention is not particularly limited, as long as the kit includes a section that contains an agent-encapsulating liposome and a section that contains a serum-free isotonic solution containing Fe ions or La ions at a concentration of from 100 μΜ to 3000 μΜ. The kit may be, for example, a container having at least these two sections. The container may further have at least section that contains a reagent that is required immediately before use and other necessary reagents. The scope of the kit for incorporating an agent into a target cell in the invention also encompasses a configuration in which each section is provided at a different container.

[0051] In the kit for incorporating an agent into a target cell in the invention, the

agent-encapsulating liposome may be in the state of being suspended in a pH buffer solution, or in the state of being freeze-dried.

The kit for incorporating an agent into a target cell in the invention may further include an instruction manual for the use of the kit. In the invention, the instruction manual for the use of the kit may be, for example, a manual that describes a method of delivering an agent into a target cell by using a serum-free isotonic solution that has a pH of from 5 to 9 and contains Fe³⁺ or La³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ.

EXAMPLES

[0052] Examples of the invention are described below. However, the invention is not limited by the examples. In the descriptions below, "%" is based on mass unless indicated otherwise.

The materials and methods employed in Examples 1 to 5 are as follows.

[0053] [Materials]

L-a-phosphatidylcholine type XVI-E (Egg-PC), dicetylphosphate (DCP), cholesterol, a-tocopherol, calcein, cytochalasin D, z-D-Phe-Phe-Gly, and propidium iodide were purchased from Sigma-Aldrich Co., (St. Louis, MO, USA).

L-a-phosphatidylethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (Rho-PE) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Chlorpromazine,

l-O-n-octyl-P-D-glucopyranoside, ampicillin sodium salt, penicillin-streptomycin mixed solution, Dulbecco's Modified Eagle Medium (DMEM), 0.5% trypan blue stain solution were purchased from Nakalai Tesque, Inc. (Kyoto, Japan). The plasmid gene, pEGFP-Nl Vector, was purchased from Clontech-Takara Bio Inc. (Shiga, Japan). DOTAP Liposomal

Transfection Reagent was obtained from Roche Applied Science (Penzberg, Germany). Dialysis tubing was purchased from Sanko Junyaku Co., Ltd (Tokyo, Japan). All the other chemicals were purchased from Kanto Chemical Co. Inc. (Tokyo, Japan) and were of the highest available purity. Deionized distilled water (Millipore, MA, USA) was used to prepare aqueous solutions.

[0054] [Cell lines and cell culture]

A mouse mammary tumor cell line (4T1) which had been maintained in RPMI 1640 medium containing 10% FBS, 0.03% kanamycin and 0.1% ampicillin was used. HeLa, HEK293, B16BL6, RAW246.7 and COS-7 cells were cultured in DMEM supplemented with 10% FCS, 25 IU/mL penicillin, 25 μg/mL streptomycin at 5% C0₂, 37°C.

[0055] [Preparation of liposomes]

Liposomes were composed of egg yolk phosphatidylcholine (Egg-PC), cholesterol (Choi), dicetyl-phosphate (DCP) and a-tocopherol. The single unilamellar vesicles (SUV) having a mean diameter about 100-200 nm were prepared from multilamellar vesicles (MLV) using a French press (FA-078A, Thermo Fisher Inc., USA). Liposomes were fluorescently labeled by encapsulating calcein solution in them at a quenching concentration of 63 mmol/L. The pH and osmotic pressure of calcein solution were adjusted to 7.4 and 308 mOsm, respectively. In some experiments, the liposomal membrane was labeled with Rho-PE. Liposome solution was then put into a cellulose tube and dialyzed to remove unenclosed calcein. The size distribution of SUV was measured in a Zeta Sizer, Model 3000HSA (Sysmex, Kobe, Japan).

[0056] EPC was used since the liposomal membrane composed of EPC was in a liquid crystal state at room temperature (phase transition temperature of EPC was from -5 to -7°C) and because of its high biological safety. Since cholesterol is known to decrease membrane fluidity, it was used in order to prevent the release of entrapped marker and to increase liposomal membrane stability. DCP or SA was added to give a negative charge or positive charge to the surface of liposomes in order to prevent their aggregation. A small amount of alpha-tocopherol was added to all liposomes except for the liposomes entrapping GFP plasmid genes in order to prevent the peroxidation of lipid.

[0057] The composition, encapsulated materials, encapsulation efficiency, mean particle size and zeta potential of the liposomes prepared in this study and indicated in Figs. 1 to 6 are shown in Table 1. 58] Table 1

[0059] [Measurement Method]

The measurement methods and confirmation test methods employed in the Examples are as described below.

[FACS analysis]

After the preincubation in the presence of metal ions, cells were incubated at 37°C for 60 min, washed twice with PBS, then resuspended in RPMI(-) containing 30mmol/L propidium iodide. The uptake of liposomes by viable 4T1 cells was measured by a fluorescence-activated cell sorting method using a BD FACSAria cell-sorter (Becton

Dickinson, NJ, USA).

[0060] [Confocal laser scanning microscopy]

Cells were washed with PBS and immobilized with 10% formalin. The intercellular localization of liposomes which were labeled both with calcein and Rho-PE was examined by a confocal laser scanning microscope (FV 1000, Olympus, Tokyo, Japan).

[0061] [Transfection of GFP gene using Fe(3+) ions]

The plasmid (pEGFP) gene was amplified in E. coli under ampicillin selection and purified using Invitrogen Miniprep columns following the manufacturer's instructions. The pEGFP-enclosing liposomes were prepared by the detergent removal method using octylglucopyranoside. The sizing of SUV was performed using a Mini-Extruder (Avanti Polar Lipids, Inc., Alabaster, Ala, USA). Unenclosed pEGFP gene was removed by gel filtration using a Sepharose 4B column. The DNA concentration was assayed using a Nano Drop 2000 (Thermo Fisher Scientific Inc., Wilmington, DE, USA) after lysis of liposomes by deoxycholic acid. From the DNA concentration, the percentage of enclosed pEGFP gene was calculated. The COS-7 cells were suspended in DMEM medium containing FBS (DMEM(+)) (cell concentration: 3 ^χ 10⁵ cells/mL). The cell suspension (1 mL) and liposome solution containing 2.5 μg DNA were added to each well, then 100 μΐ, of 20 mmol/L Fe(3+)solution (final concentration: 1 mmol/L) and DMEM(+) were added to each well of a 6-well plate so that total volume was 2 mL. After 24 hours, fluorescence intensity of cells was measured by FACS and the percentage of GFP-expressing cells compared to total cells was calculated. Fluorescence microscopic images were also observed.

[0062] <Example 1> The dependency of uptake on the Fe (3+) concentration

The 4T1 cells were suspended in RPMI medium containing no FBS (RPMI(-)) (cell concentration: 1.5-2 ^χ 10⁶ cells/mL). The cell suspension (450 μί) and 50 \L of liposome solution (amount of lipids: 260 μg) were added to a cuvette and mixed.

Further, Fe³⁺ ions were added such that the final concentration of Fe³⁺ ions became 0 (designated by "control" in the figure), 100 μΜ, 300 μΜ, 500 μΜ, 800 μΜ, and 1000 μΜ. The vertical axis in Fig. 1 represents a ratio when the value of the "control" is set to 1.

[0063] The results are shown in Fig. 1.

The results clarified that addition of Fe ions at from 100 to 1000 μιηοΙ/L promotes uptake of the calcein-encapsulating liposome into the target cells.

Particularly, it was further clarified that addition of Fe³⁺ ions at a concentration as high as 300 μπιοΙ/L further promotes the effect.

[0064] <Example 2> Study of uptake mechanism by inhibitors and CLSM

In order to examine the uptake-inducing mechanism of Fe(3+) ions, the influence of various inhibitors was investigated. The concentration of inhibitors and time of exposure were determined from the condition at which cell viability was more than 90% and the inhibition was maximal. It is known that cytochalasin D (CytD) inhibits phagocytosis and macropinocytosis(Methods Enzymol(1983) 98:368-375), chlorpromazine (CPZ) inhibits clathrin-mediated endocytosis (J Cell Biol (1993) 123:1107-117, and Z-D-Phe-Phe-Gly (ZfFG) inhibits membrane fusion (Arch Biochem Biophys (2003) 410:246-253).

[0065] The results are shown in Fig. 2.

The uptake of liposomes by 4T1 cells was inhibited by CytD (about 50%) or CPZ (about 67%) but was not inhibited by ZfFG in the absence of Fe(3+) ions. Uptake was inhibited only by ZfFG (about 43%) in the presence of Fe(3+) ions however.

Confocal laser scanning microscopy (CLSM) images are shown in Fig.3.

[0066] The liposome lipid membrane and the inner aqueous phase were labeled with Rho-PE (red fluorescence) and with calceine (green fluorescence), respectively. The red and green fluorescence localized at almost the same intracellular position in the absence of Fe(3+) ions, showing that the liposomes were internalized by endocytosis and localized in a phagosome or phagolysosome without being lysed. Red fluorescence was localized on the cell membrane surface and the whole inside of the cell was dotted with green fluorescence in the presence of Fe(3+) ions.

This observation showed that liposomes fused with cell membrane and the enclosed calcein diffused into the cytoplasm.

Considering the findings obtained both from the inhibition study and the CLSM observation, it was suggested that the uptake of liposomes by 4T1 cells in the presence of Fe(3+) ions took place via membrane fusion.

[0067] <Example 3> Uptake of liposomes by various cell lines

The delivery efficiency of the calcein-encapsulating liposome into the other cell lines was studied using the same method as in the case of a Fe³⁺ ion concentration of 1000 μπιοΙ/L in Example 1. The results are shown in Fig. 4.

The uptake-inducing effect of Fe(3+) ions which was observed using 4T1 cells was also tested using the other cell lines, that is, B 16BL6, HeLa, RAW and HEK293.

All four cell lines exhibited about 10-18-fold the inducing effect compared to the control, suggesting that the uptake-inducing effect of Fe(3+) ions is applicable to various cell lines.

[0068] <Example 4> Effect of Fe(3+) ions concentration on GFP gene expression and cell viability

The effect of Fe(3+) ion concentration and cell viability were assayed according to the method described in "[Transfection of GFP gene using Fe(3+) ions]" in "[Measurement Method]" above.

[0069] The results are shown in Fig. 5.

The effect of Fe(3+) ion concentration on both GFP expression and cell viability was then investigated. About 80% cell viability and 32% GFP-expressing cells were obtained even at 1000 μιηοΙ/L and no marked cytotoxicity was observed.

These results clarified that the problem of cytotoxicity does not occur even when the concentration of Fe³⁺ ions are increased to be as high as 1000 μΜ in order to increase the delivery efficiency of the agent into the target cell.

[0070] <Example 5> Comparison with commercial gene transfection reagents

The transfection of pEGFP into COS-7 cells was performed under the optimal conditions mentioned above. As a comparison, transfection of pEGFP was performed using DOTAP Liposomal Transfection Reagent. The amount of pEGFP and the number of cells in both experiments were identical. DOTAP was used according to the directions for use.

[0071] The results are shown in Fig. 6.

The expression of GFP was barely observed in the control, while the percentage of GFP-expressing cells was about 22% when Fe(3+) ions were used (Fig.6)

This value was significantly higher than the expression rate (16%) when DOTAP reagent was used. A similar result was also obtained when using CLSM (data not shown).

[0072] The result clarified that the method of delivering an agent into a target cell according to the invention is superior to conventional methods.

The materials used in Examples 6 to 10 are as described below.

[0073] [Cell culture]

A mouse mammary tumor cell line (4T1) which had been maintained in RPMI 1640 medium containing 10% FBS, 0.03% kanamycin and 0.1% ampicillin was used.

African green monkey SV-40 transfected kidney fibloblast cell line (Cos-7) were cultured in DMEM supplemented with 10% FCS, 25 IU/mL penicillin, 25μ^πιΙ, streptomycin at 5% C0₂, 37°C. The 70-80% confluent adherent 4T1 cells were washed twice with lOmL of PBS solution. Then 1.5 mL of 0.05% trypsin solution containing 0.53 mmol/L EDTA was added and the cells were incubated for 5 min at 5% C0₂, 37°C. The trypsin activity was inhibited by adding 10 mL RPMI(+) medium and the solution containing non-adherent cells was centrifuged at 200 ^χ g for 5 min. Cell suspension was obtained by adding 10 mL of

RPMI(+) to the cell precipitation. The cell viability was assayed using 0.5% trypan blue solution.

[0074] [Preparation of liposomes]

(Preparation of multilamellar vesicles (MLV))

Egg PC (10 mg), Choi, (0.279 mg) and DCP (0.395 mg) were dissolved in 3 ml of chloroform in an ice bath, and the solution was vortexed under nitrogen atmosphere. The organic solvent was evaporated in a rotary evaporator until a thin lipid film was formed. The film was dried for 8 h under vacuum. Then 2ml of 63 mmol/L calcein aqueous solution (pH 7.4, 308 mOsm/L) was added to the lipid film and the solution was vortexed for 20 min under nitrogen atmosphere.

[0075] (Preparation of single unilamellar vesicles (SUV))

The SUV having a mean diameter about 100 -200 nm were prepared from MLV using a Mimi Extruder (Avanti Polar Lipids, Inc., Alabama, USA).

Unenclosed calcein was removed by gel filtration (Sepharose 4B) using aqueous sucrose solution (102.7g/L) containing HEPES (1.2 mg/L) as the eluent. The size

distribution of SUV was measured in a Zeta Sizer, Model 3000HSA (Sysmex, Kobe, Japan).

[0076] <Example 6>

Uptake of calcein-encapsulating liposomes by 4T1 cells in the presence of Fe(3+) or La(3+) ions

The 70-80% confluent 4T1 cells were treated by trypsin (concn.: 0.1 mg/mL) and washed twice with PBS, then resuspended in RPMI(-) medium (concn. of cells was 1.5 - 2 ^χ 10⁶ cells/mL). After 25 SUVsolution (amount of lipids: 130 μg) was added to 200 μΐ, of cell suspension, 25 μΐ, of Fe(3+) or La(3+) solution (final concn. of metal ion was 1 x 10^" mol/L) was added and the mixture was incubated at 37°C for 60 min. The cells were washed twice with 1 mL PBS, then suspended in 300 μί_^ of RPMI(-) medium.

The uptake of liposomes by viable 4T1 cells was measured by a

fluorescence-activated cell sorting method using a BD FACSAria cell-sorter.

[0077] The results are shown in Fig. 7.

The results clarified that both of Fe³⁺ ion and La³⁺ ion are capable of delivering an agent into a target cell.

In particular, it was clarified that use of Fe³⁺ ions allows delivery of an agent into a target cell with higher efficiency as compared to use ofLa³⁺ ions.

[0078] <Example 7> Uptake of calcein-encapsulating SUV by suspended 4T1 cells in the presence and in the absence of FBS.

The 70-80% confluent 4T1 cells were treated by trypsin (concn. was 0.1 mg/mL) and washed twice with PBS, then resuspended in RPMI(+) or RPMI(-) medium (concn. of cells was 1.5-2 x 10⁶ cells/mL). After 25 μΐ, liposome solution (amount of lipids: 130 μg) was added to 200 μΐ, of cell suspension, 25 μί of Fe(3+) solution (final concn. of metal ion was 1 x 10^~3 mol/L) was added and the mixture was incubated at 37°C for 60 min. The cells were washed twice with 1 mL PBS, then suspended in 300 ΐ. of RPMI(-) medium.

The uptake of SUVby 4T1 cells was measured as described in Example 6 above.

[0079] The results are shown in Fig. 8.

The results clarified that, in order to efficiently deliver an agent into a target cell when the target cell is in the suspended state, not only addition of Fe³⁺ ions, but also use of a serum-free medium, is necessary.

[0080] <Example 8> Uptake of calcein-encapsulating liposomes by adherent 4T1 cells in the presence and in the absence of FBS

The uptake of liposomes by 4T1 cells was measured as described in Example 6 above.

The results are shown in Fig. 9.

The results clarified that, in order to efficiently deliver an agent into a target cell when the target cell is in the adhered state, not only addition of Fe³⁺ ions, but also use of a serum-free medium, is necessary.

[0081] <Example 9> Uptake-inducing effect of Fe(3+) ions

[Uptake of SUV by 4T1 cells]

(Uptake of SUV in the presence -of Fe(3+))

The 4T1 cells were suspended in sucrose solution (1.5-2 ^χ 10⁶ cells/mL). The cell suspension (250 μί,) and 100 \L of SUV solution (amount of lipids: 140 μg) were added to a sampling tube and mixed. Then 100 μΐ, of FeCl₃ dissolved in PBS (final concn.: 1000 μιηοΙ/L) was added and the mixture was incubated for 60 min. The 4T1 cells were washed twice with PBS and re-suspended in 300 μΐ, RPMI(-) medium. The uptake of SUV by viable 4T1 cells was measured using a BD FACSAria cell-sorter (Becton Dickinson, NJ, USA).

[0082] The results are shown in Fig. 10.

The uptake of the calcein-encapsulating liposome by 4T1 was promoted in the presence of Fe³⁺ ions as compared to in the absence of Fe³⁺ ions.

[0093] <Example 10> Transfection of GFP gene using Fe(3+) ions

[Preparation of GFP gene]

The plasmid gene, pEGFP-Nl vector was purchased from Clontech-Takara Bio Inc. (Shiga, Japan). The pEGFP gene was amplified in E.coli under ampicillin selection and purified using Invitrogen Midiprep columns following the manufacturer's instructions.

[Preparation of GFP gene-entrapping SUV]

The GFP gene-entrapping SUV were prepared as described in "[Preparation of liposomes]" of Example 6 using a GFP gene solution which contained 17.5 mg NaCl, 2.4 mg HEPES and 200 μg pEGFP in 2 mL water instead of calcein solution. The DNA

concentration was assayed using a Nano Drop 2000 (Themo Fisher Scientific Inc.,

Wilmington, DE, USA) after lysis of SUV by 100 mmol/L sodium deoxycholic acid. From the DNA concentration, the percentage of enclosed GFP gene was calculated.

[0084] [Transfection of GFP gene]

The 4T1 cells or Cos-7 cells were suspended in sucrose solution (cell concentration was 2 x 10⁶ and 5 ^χ 10⁵ cells/mL, respectively). The cell suspension (250 μί,) and SUV solution containing 2.5 μg DNA were added to each well, then FeCl₃ solution dissolved in either PBS or HEPES (final concentration : 10-2000 μηιοΙ/L) and sucrose solution were added so that total volume was 0.5mL. After incubating at 37°C for lh, the cells were washed with PBS and cultured in RPMI(+) or DMEM(+). The cells were treated with trypsin solution after 24 h and fluorescence intensity of cells was measured by FACS and the percentage of GFP-expressing cells compared to total cells was calculated.

[0085] [Optimization of transfection condition]

To optimize GFP gene-transfection condition, effects of composition and pH of buffer solution which dissolved FeCl₃ and of Fe(3+) concentration were examined. The PBS (pH6.0) contained 8g NaCl, 0.2g KCl, 1.12g KH₂P0₄, 0.35g Na₂HP0₄-7H₂0 in 1L H₂0, PBS(pH7.4) contained 8g NaCl, 0.2g KCl, 0.2g KH₂P0₄, 2.17g Na₂HP0₄-7H₂0 in 1L H₂0 and PBS(pH8.0) contained 8g NaCl, 0.2g KCl, 0.07g KH₂P0₄, 2.41g Na₂HP0₄-7H₂0 in 1L H₂0. The HEPES contained 8.8g NaCl and 1.2g HEPES in 1 L H₂0.

[0086] The results are shown in Figs. 11 to 16.

The results shown in Figs. 11 and 12 clarified that the method of delivering an agent into a target cell according to the invention is superior to conventional methods, not only in 4T1 cells but also in Cos-7 cells.

[0087] The results shown in Fig. 13 clarified that the pH of PBS in the invention is most preferably from pH6.0 to pH7.4. Here, the control represents the percentage of GFP-expressing cells observed when the cells and SUV were incubated in a 102.7g/L sucrose aqueous solution containing 1.2mg/L HEPES.

The results shown in Fig. 14 clarified that Fe³⁺ ions exerts effect in terms of promoting uptake of an agent-encapsulating liposome into a target cell, irrespective of the presence or absence of phosphoric acid.

[0088] The results shown in Fig. 15 clarified that the effect of the invention cannot be obtained when the concentration of Fe ions is as low as 10 μΜ.

The results shown in Fig. 16 clarified that the uptake of an agent-encapsulating liposome into a target cell in the absence of phosphoric acid is promoted by addition of Fe ions at a concentration as high as 1 mM or higher.

[0089] It was suggested that the uptake of liposomes by 4T1 cells in the presence of Fe(3+) ions took place via membrane fusion. The transfection of pEGFP gene into COS-7 cells can be efficiently performed by means of Fe(3+) ions. The findings obtained in this study that Fe(3+) ions promoted the cell fusion of liposomes will give a useful knowledge for developing a new gene transfection method by fusion.

[0090] According to the invention, a method of efficiently delivering an agent encapsulated in a liposome into a target cell can be provided.

The present application claims the benefits of priority to U.S. provisional application Ser. No. 61/256,336, filed Oct 30, 2009. The contents of U.S. provisional application Ser. No. 61/256,336 are incorporated herein by reference in their entirety. All publications, patent applications, and technical standards mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A method of delivering an agent into a target cell, comprising contacting an agent-encapsulating liposome with the target cell in a serum-free isotonic solution that has a pH of from 5 to 9 and contains Fe³⁺ or La³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ, so as to deliver the agent into the target cell.

2. The method of delivering an agent into a target cell according to claim 1, wherein the serum-free isotonic solution is a phosphate buffer solution that has a pH of from 5 to 9 and contains Fe³⁺ or La³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ.

3. The method of delivering an agent into a target cell according to claim 1, wherein the serum-free isotonic solution is a Good's buffer that has a pH of from 5 to 9 and contains Fe³⁺ or La³⁺ ions at a concentration of from 500 μΜ to 3000 μΜ.

4. The method of delivering an agent into a target cell according to claim 3, wherein the Good's buffer is a HEPES solution.

5. The method of delivering an agent into a target cell according to any one of claims 1 to 4, wherein the serum-free isotonic solution is a serum-free isotonic solution that has a pH of from 5 to 9 and contains Fe³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ.

6. The method of delivering an agent into a target cell according to any one of claims 1 to 5, wherein the agent is of at least one type selected from the group consisting of a nucleic acid, a drug, a chemical substance and a protein.

7. The method of delivering an agent into a target cell according to claim 6, wherein the drug is an anticancer agent.

8. The method of delivering an agent into a target cell according to claim 6, wherein the chemical substance is a differentiation inducing agent or a reprogramming agent.

9. The method of delivering an agent into a target cell according to any one of claims 1 to 8, wherein the target cell is of at least one type selected from the group consisting of a cancer cell, a tissue-derived cell, an organ-derived cell, and a stem cell.

10. The method of delivering an agent into a target cell according to any one of claims 1 to 9, wherein the target cell is a cancer cell, and the agent is an anticancer agent.

11. The method of delivering an agent into a target cell according to any one of claims 1 to 9, wherein the target cell is a stem cell, and the agent is a differentiation inducing agent.

12. The method of delivering an agent into a target cell according to any one of claims 1 to 9, wherein the target cell is a tissue-derived cell or an organ-derived cell, and the agent is a reprogramming agent.

13. A serum-free isotonic solution containing Fe³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ and having a pH of from 5 to 9.

14. The serum-free isotonic solution according to claim 13, wherein the serum-free isotonic solution is a phosphate buffer solution containing Fe³⁺ ions at a concentration of from 100 μΜ to 3000 μΜ and having a pH of from 5 to 9.

15. The serum-free isotonic solution according to claim 13, wherein the serum-free isotonic solution is a Good's buffer containing Fe ions at a concentration of from 500 μΜ to 3000 μΜ and having a pH of from 5 to 9.

16. The serum-free isotonic solution according to claim 15, wherein the Good's buffer is a HEPES solution.

17. The serum- free isotonic solution according to any one of claims 13 to 16, wherein the serum-free isotonic solution is used for delivering an agent into a target cell.

18. A pharmaceutical composition comprising the serum-free isotonic solution of claim 13 and an agent-encapsulating liposome.

19. An agent-delivering kit comprising a section including the serum-free isotonic solution of claim 13 and a section including an agent-encapsulating liposome.