JP2001335512A - Microparticle for transducing gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a carrier for an agent, a physiologically active substance, a gene or the like at low cost, having a high introduction or transduction efficiency of a useful substance and preparable by simple operations. SOLUTION: This microparticle for a pharmaceutical preparation is characterized as comprising a phospholipid, a cationic lipid and a polyethylene glycol binding lipid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to fine particles for pharmaceutical preparations which can introduce useful substances such as drugs, physiologically active substances, and genes into cells of a target tissue with high efficiency. The present invention relates to a conjugated gene transfer agent.

[0002]

2. Description of the Related Art When treating a disease, a carrier is required for introducing useful substances such as drugs and physiologically active substances (hormones, lymphokines, etc.) into cells of a target tissue. In particular, in recent years, with the advance of technologies such as gene therapy and antisense drugs, carriers for efficiently introducing a specific gene into cells of a target tissue have been actively developed.

Hitherto, as a carrier for introducing a specific gene into cells of a target tissue, a viral vector has been used (Hodgson, Bio / Technology 3,222 (1992)).
5), Jolly, Cancer Gene Therapy 1, 51 (1994), etc.) For example, there is a risk that a wild-type virus having a growth ability may appear due to unexpected homologous recombination between a virus vector and a wild-type virus in cells. , There was a problem. Therefore, a method using a liposome as a non-viral carrier has been proposed.

A liposome is an endoplasmic reticulum formed when a lipid is dispersed in water and having an inner aqueous phase closed by a lipid bilayer, and has a basic structure similar to the plasma membrane of a living cell. In addition to its high affinity with biological membranes, viruses can be encapsulated inside lipid bilayer membranes or in the internal aqueous phase, and are easily bio-metabolized and have low toxicity and immunogenicity. It has various advantages not found in vectors and can be expected to be used as a carrier.

In practice, a gene encoding a physiologically active substance is administered to a patient in a state of being encapsulated and held in the inner aqueous phase of the liposome or as a complex with the liposome to introduce the gene into cells of the target tissue. Also, gene therapy for producing a physiologically active substance has been studied. Examples of such a gene carrier include DNA having a negative charge.
Cationic liposomes prepared from cationic lipids that can easily form a complex with liposome are used (JP-A-9-278726).
JP, JP-A-11-187873, etc.).

[0006]

However, conventional cationic liposomes have a problem in that the efficiency of gene transfer into cells of a target tissue is low. Although the cationic liposome has a large positive charge of the lipid itself, the positive charge is oriented not only on the outer side of the liposome but also on the inner aqueous phase side because of the lipid bilayer structure. That is, since the positive charge of the lipid is not effectively used for forming a complex with DNA, there is a problem that the rate of forming a complex with DNA is low and the efficiency of introducing a gene into cells of a target tissue is low. .

[0007] In addition, cationic liposomes cannot be used immediately, and there is a problem that a long time is required for preparation and preparation of the cationic liposome (eg, 9 steps, 2 days). The cationic lipid constituting the cationic liposome often has an ester bond, and is easily susceptible to hydrolysis by an acid or base catalyst. Therefore, cationic liposomes are unstable in a suspension state and are often stored in a frozen state. Therefore, pretreatment such as freezing and thawing is essential for use. That is, it cannot be said that it can be prepared by a simple operation, and there is a problem that its preparation and preparation require a long time.

[0008] Furthermore, cationic liposomes have the problem that they are extremely expensive in terms of cost due to the use of synthetic lipids having a special structure.

Therefore, the conventional cationic liposome is
Although excellent effects can be expected as a carrier for introducing useful substances such as drugs, physiologically active substances, and genes into cells of target tissues, at present, in terms of gene transfer efficiency, simplicity and cost of preparation, However, it is not satisfactory in terms of the above.

[0010] That is, an object of the present invention is to provide a carrier for a drug, a physiologically active substance, a gene, or the like, which has a high efficiency of introducing a useful substance, can be prepared by a simple operation, and is low in cost.

[0011]

Means for Solving the Problems As a result of intensive studies by the present inventors, the above problems of the prior art can be solved by using fine particles containing phospholipid, cationic lipid and polyethylene glycol-bound lipid as a carrier. The inventors have found out what can be done and completed the present invention.

That is, the present invention relates to pharmaceutical microparticles containing a phospholipid, a cationic lipid, and a polyethylene glycol-bound lipid.

[0013] The present invention also relates to a gene transfer agent, which is a complex of the above-mentioned fine particles for preparation and a gene.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the fine particles for pharmaceutical preparation of the present invention (hereinafter, simply referred to as “fine particles”) will be described in detail.

(1) Constituents The phospholipid used in the present invention is pharmaceutically acceptable.
Although it is not particularly limited as long as it can be a base constituting the fine particles, it is preferable to use a natural phospholipid in terms of low toxicity to living organisms, easy availability and low cost, and the positive charge of the cationic lipid described later. Is more preferably a phospholipid having a charge that does not cancel

Examples of the phospholipid include natural phospholipids such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean lecithin, lysolecithin and the like. (For example, hydrogenated soybean lecithin) and the like. Among them, soybean lecithin is preferably used because it has been used as an injection. In this specification, the term “phospholipid” does not include a cationic lipid and a polyethylene glycol-bound lipid described below.

The type of the acyl group constituting the hydrophobic portion of the phospholipid is not particularly limited, and examples thereof include an acyl group such as a lauroyl group, a myristoyl group, a palmitoyl group, a stearoyl group, and an oleoyl group.

As long as the cationic lipid used in the present invention is a lipid having a positively charged atomic group such as quaternary ammonium in the molecule, the structure of the lipid portion, the type of the positively charged atomic group, and the like are as follows. There is no particular limitation. By including a cationic lipid as a component, a positive charge is imparted to the outer surface of the fine particles, and a negatively charged gene (DN
A) (the nucleic acid such as A).

Such cationic lipids include, for example, 3
β [N- (N7, N′-dimethylamino-ethane) -carbamoyl] cholesterol (hereinafter referred to as “DC-cholesterol”), stearylamine, N- (α-trimethylammonioacetyl) dodecyl-D-glutamate chloride , N- [1- (2,3-oleoyloxy)
Propyl] -N, N, N-trimethylammonium chloride, 2,3-dioleoyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneammonium trifluoroacetate and the like No. Among them, DC-cholesterol and / or stearylamine are preferable in that they are inexpensive and easily available.

The polyethylene glycol-bound lipid (hereinafter referred to as “PEG lipid”) used in the present invention is not particularly limited as long as it is pharmaceutically acceptable and a lipid having a PEG chain bound thereto. Is the natural phospholipid described above, and the PEG chain has a weight average molecular weight of 1
About 000 to 12000 is preferable.

The inclusion of PEG lipid as a component enhances the interaction between the microparticle and the cell, and promotes the fusion between the microparticle and the cell. In addition, when PEG lipids are contained, the PEG chains are oriented to the outside of the cells, and the fine particles are not trapped in the reticuloendothelial system such as the liver. It also has the effect of increasing the half-life.

As such a PEG lipid, polyethylene glycol 2000-distearoyl phosphatidylethanolamine (hereinafter referred to as "PEG2000-DSPE") is preferable because it has been used in liposome preparations.

The above phospholipid, cationic lipid, PEG
Any of the lipids can be used alone or in combination of two or more. The ratio of each lipid in the above constituent components was 76.4 to 12% by mass of phospholipid, 11.5 to 80% by mass of cationic lipid, and 5% of PEG lipid.
It is preferably about 40% by mass.

When the microparticles of the present invention are used as a gene transfer agent, calcium chloride and / or glycerin may be further added to enhance the interaction with cells or the effect of transferring genes into cells, in addition to the above-mentioned components. Etc. may be contained. The effect is exhibited by adding 1% by mass or less of calcium chloride and 0.01 to 2% by mass of glycerin with respect to the whole of the above components.

As described above, the fine particles of the present invention
Preferably, the phospholipid is soy lecithin and the cationic lipid is 3β [N- (N7, N'-dimethylamino-
[Ethane] -carbamoyl] cholesterol and / or stearylamine, the polyethylene glycol-bound lipid is preferably polyethylene glycol-bound distearoyl phosphatidylethanolamine, and preferably contains calcium chloride and / or glycerin.

(2) Method for Preparing Fine Particles The fine particles of the present invention can be prepared, for example, by the following method.

First, phospholipid, cationic lipid, and P
The EG lipid is dissolved in a suitable solvent and mixed. Mixing is performed using water, various organic solvents (for example, lower alcohols such as methanol and ethanol, chloroform and the like), a mixture thereof, or the like at room temperature or at 40 to 80.
It can be carried out under heating at ° C.

Next, the solvent of the above mixed solution is distilled off and concentrated. As a method for distilling off the solvent, a rotary evaporator, a vacuum dryer or the like is used at room temperature or at 40 ° C.
The heating can be performed at a temperature of from about 80 ° C. to 80 ° C., and is preferably performed under an N ₂ gas stream.

After distilling off the solvent, water and, if desired, an aqueous solution containing calcium chloride and / or glycerin are added, and the lipid is dispersed in water by high-speed stirring using a stirrer such as a homogenizer. I do.
The fine particles of the present invention preferably have a particle size of 200 μm or less in order to ensure the function. Examples of a method for obtaining such fine particles include a method in which the stirring speed is as high as possible (for example, 23000 rpm), a method in which a filter is sized, and the like.

The stirring is preferably performed under ice cooling in order to suppress the oxidation of lipids due to a rise in the temperature of the solution, and specifically, a method of stirring with a homogenizer while cooling with ice water may be mentioned. The sizing and sterilization can be performed at the same time by filtration using a membrane filter having a pore size of 0.45 μm or less at room temperature in a clean bench.

The fine particles prepared as described above are characterized in that they are nanoparticles that do not have a lipid bilayer structure like liposomes and do not have an internal aqueous phase. That is, since all the positive charges are oriented to the outside of the microparticles and are effectively used for forming a complex with DNA, the rate of forming a complex with DNA is high, and the efficiency of gene transfer into cells of the target tissue is improved. It is possible to do. In addition, since it is stable in an aqueous solution and does not require pretreatment such as freezing and thawing like a conventional cationic liposome, it can be easily prepared in a short time, and can be used immediately.

(3) Formulation Method The microparticles of the present invention can be easily prepared as described above, and can be easily mixed with a useful substance to be introduced into cells of a target tissue, and can be easily complexed with each other. Can be formed and formulated. That is, the preparation and preparation of the microparticles can be performed in a very simple operation in a single step and in a short time of about 30 minutes, so that they can be particularly suitably used as a gene transfer agent combined with a gene. .

[0033]

As described above, since the fine particles of the present invention are nanoparticles composed mainly of phospholipids, they have a high rate of complex formation with DNA, and can transfer genes into cells of target tissues. It is possible to improve the introduction efficiency. In addition, since it is stable in an aqueous solution and does not require pretreatment such as freezing and thawing like a conventional cationic liposome, it can be easily prepared in a short time, and can be used immediately. Furthermore, since it can be prepared using an easily available and inexpensive lipid, there is no problem in terms of cost. Therefore,
The microparticles of the present invention are particularly useful as a gene carrier that replaces conventional cationic liposomes, which were not sufficient in terms of gene transfer efficiency or in terms of simplicity and cost of preparation.

[0034]

The present invention will now be described in more detail with reference to examples of a gene transfer agent in which the fine particles of the present invention are combined with a gene. However, the present invention is not limited to the following examples.

Example 1-1 In Example 1-1, soybean lecithin (manufactured by Fuda Pharmaceutical Co., Shanghai, China) was used as a phospholipid, and DC-cholesterol (manufactured by Sigma) and stearylamine were used as a cationic lipid. (Manufactured by Sigma) as PEG lipid using PEG2000-DSPE
Using Nippon Yushi Co., Ltd., fine particles having the composition ratios shown in Table 1 were prepared.

[0036]

[Table 1]

First, while heating at about 45 ° C., 100 mg of soybean lecithin, 10 mg of DC-cholesterol, 5 mg of stearylamine, and 15 mg of PEG2000-DSPE are dissolved in 14 ml of ethanol (manufactured by Wako), and the resulting mixture is rotated with a rotary evaporator (Tokyo Rika Chemical Co., Ltd.). Ethanol was distilled off using a vacuum dryer (Model: VO-Z-4 / Shimizu Rikagaku Kikai Seisakusho) until the solution volume was about 0.1 ml, and concentrated.

Next, 0.01 g / 1 was added to the lipid thin film.
00 ml of calcium chloride, and 0.15 g / 100 m
1 ml of an aqueous solution containing glycerin, and add a total of 1
0 ml and 23000 rpm using a homogenizer.
For 6 minutes to make the lipid fine particles.

Further, the particles were filtered in a clean bench at room temperature using a membrane filter having a pore size of 100 nm (trade name: Nikulipa Membrane, manufactured by Nomura Science Co., Ltd.), and subjected to particle size control and sterilization treatment. A liquid was prepared. The microparticle liquid was sealed in a 10 ml ampule. The time required to prepare the fine particle liquid of Example 1-1 was about 30 minutes.

Comparative Example 1-1 In Comparative Example 1-1, the cationic liposome reagent Tfx-
20 (manufactured by Promega) was suspended in water, heated at 65 ° C. for 1 minute, frozen at −20 ° C. overnight, and then thawed to prepare a liposome. The time required to prepare the liposome solution of Comparative Example 1-1 was about 12 hours.

Fine particles of Example 1-1, Comparative Example 1-1
For the liposome, the gene was compounded into a gene transfer agent, and the performance was evaluated based on the efficiency of gene transfer into target cells.

Specifically, after the target cells were brought into contact with the above-mentioned gene transfer agent and cultured for 24 hours, the culture solution was illuminated with a luciferase enzyme kit, and the efficiency of gene transfer was evaluated by measuring the fluorescence intensity. (Luciferase assay). In addition, the luciferase activity value was evaluated by converting into a luciferase activity value per 1 fg of protein.

In the above luciferase assay, a luciferase enzyme kit was manufactured by Promega, and a chemiluminometer (trade name: MicroLumat LB96P, manufactured by EG & G Berthold) was used as a fluorescence intensity measuring device. The protein was quantified using a CBB reagent (trade name: CBB Dye Reagent (5-fold concentrated) / Nacalai Tesque, Inc.).

Genes to be complexed with microparticles or liposomes include the name of research institute, City University of New York, M
Plasmid DNA (AAV-CMV-Luc) provided by Dr. Charless Mobbs of t.Sinai Medical University was used (Sugiyama A., Hattori S., Tanaka S., Isoda F., Kleop).
oulos S., Kaplitt M., Sekihara H., Mobbs C.:Defective
adenoassociated viral-mediated transfection of in
sulin gene by directinjection into liver parenchym
a decreases blood glucose of diabetic mice.Horm.Me
tab. Res., 29 (1997) 599-603).

On the other hand, 293 cells were used as target cells into which the gene was to be introduced. Dulbecco ME as a culture solution
M (Sales: NIPRO, Manufacturing: Kojin Bio) /
An RPMI 1640 culture solution (manufactured by Life Tech Oriental Co., Ltd.) was used (hereinafter simply referred to as "culture solution").

(Example 1-2) In Example 1-2, 8 μl of the fine particle solution of Example 1-1 and the plasmid D
A gene transfer agent was prepared by gently mixing 1 μl (2 μg) of NA and 31 μl of the above culture solution, and allowing the mixture to stand at room temperature for 10 minutes.

On the other hand, the cultured 293 cells were divided into 3 × 10 ⁵ cells / well in each well of a cell culture plate (12 wells, manufactured by Sumitomo Bakelite Co., Ltd.). The wells were washed once with an acid buffer (PBS) and once with a serum-free culture medium.
The culture solution of e was dispensed at 1 ml each.

Next, 40 μl of the prepared gene transfer agent was added to each well, and CO _{2 was} added at 37 ° C.
The gene was transferred by culturing in an incubator, and after a predetermined time, the culture solution was added to each well to terminate the gene transfer. The control was plasmid DNA
Except that no was added, the same operation was performed.

(Comparative Example 1-2) In Comparative Example 1-2, 6 µl of the liposome solution of Comparative Example 1-1, 1 µl (2 µg) of the above plasmid DNA, and 400 µl of the above culture solution were gently mixed and allowed to stand at room temperature for 10 minutes. Thus, a gene transfer agent was prepared. On the other hand, the cultured 293 cells were prepared in Example 1
-3 × 10 3 per well of the same cell culture plate as in -2
^The cells were divided into ⁵ cells / well and cultured for 24 hours.

After the culture, the culture solution is removed by suction from the cells, and
407 μl of the prepared gene transfer agent was added to each well.
After vortexing, at 37 ° C,
CO _TwoPerform gene transfer by culturing in an incubator
After a lapse of a predetermined time, the culture solution is added to each well to transfer
The child introduction was terminated.

(Results) As shown in Table 2, Example 1-1
When the microparticles of Example 1 were used, the luciferase activity per protein was clearly higher than when the liposome of Comparative Example 1-1 was used, and it was confirmed that the plasmid DNA was efficiently introduced into the cells. Was done.

[0052]

[Table 2]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/24 A61K 47/24 47/28 47/28 48/00 48/00 // C12N 15/09 C12N 15/00 A F term (reference) 4B024 AA01 AA20 CA01 EA04 GA11 HA17 4C076 AA16 CC26 DD22 DD38 DD49 DD52 EE51 FF16 FF36 FF43 GG45 4C084 AA13 AA16 BA44 MA01 NA03 NA13 ZC012 4C086 AA01 AA02 MA41 MA02

Claims (6)

[Claims] 1. Pharmaceutical microparticles comprising a phospholipid, a cationic lipid, and a polyethylene glycol-bound lipid. 2. The pharmaceutical microparticles according to claim 1, wherein the phospholipid is soy lecithin. 3. The method according to claim 2, wherein the cationic lipid is 3β [N- (N7,
N'-dimethylamino-ethane) -carbamoyl] cholesterol and / or stearylamine.
Or the fine particles for pharmaceutical preparation according to 2. 4. The fine particles for pharmaceutical preparation according to claim 1, wherein the polyethylene glycol-bound lipid is polyethylene glycol-bound distearoylphosphatidylethanolamine. 5. The fine particles for pharmaceutical preparation according to claim 1, further comprising calcium chloride and / or glycerin. 6. A gene transfer agent, which is a complex of the fine particle for preparation according to any one of claims 1 to 5 and a gene.