JP2007063194A - Material-introducing carrier, method for introducing material and kit for introducing material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material-introducing carrier based on a liposome and capable of introducing the material into a cell in high efficiency; and to provide a method for introducing the material and a kit for introducing the material. <P>SOLUTION: A composite of a mRNA-liposome-carbonate apatite particle which is a m-RNA-introducing carrier is made by dissolving a heat-denatured mRNA and DOTAP of a cationic lipid in a bicarbonate-buffered DMEM medium, incubating the dissolved product for 15 min at room temperature, adding CaCl<SB>2</SB>thereto, and further incubating the added product for 30 min at 37°C. The mRNA can be introduced highly efficiently and expressed by incubating the composite of the mRNA-liposome-carbonate apatite particle with a target cell. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a substance introduction carrier, a substance introduction method, and a substance introduction kit suitable for use in introducing drugs, physiologically active substances, DNA, RNA, or the like into cells.

  2. Description of the Related Art In recent years, in the field of medicine, development of drugs that act directly on affected areas and have high side effects with few side effects has been actively conducted. In particular, a method called a drug delivery system (DDS) is attracting attention as a method capable of specifically transporting an active ingredient such as a drug to a target cell or target tissue and allowing the active ingredient to act at a target location. .

  Also in the recent field of molecular cell biology, introduction of DNA into target cells is indispensable when analyzing the structure, function or control mechanism of genes. This technology is also extremely important in industrial production of proteins that are important in the medical field, gene therapy, or DNA vaccines.

  Various methods for introducing a drug, a physiologically active substance, a substance such as DNA into cells are known, and among them, a liposome method is a highly safe substance introduction method. This method has no risk of immune reaction unlike the viral vector method, does not damage cells like the ultrasonic method and electroporation method, and does not require special equipment, so many researchers However, there is a problem that the substance introduction efficiency into the cell is low. Therefore, in order to increase the substance introduction efficiency into the cell, an attempt has been made to add a cationic lipid having a positive charge to the liposome as a constituent component of the liposome, in addition to the neutral phospholipid which is the main component, to form a cationic liposome. Yes.

  Furthermore, attempts have been made to introduce mRNA into cells using this cationic liposome (see Non-Patent Documents 1 and 2). When DNA is introduced into cells, in order to express the protein encoded by the DNA, it is necessary for the DNA to pass through the nuclear membrane and move into the nucleus, but most target cells in vivo are nuclear. Since it is a non-dividing cell in which the membrane has not disappeared, the expression efficiency of the protein is reduced. On the other hand, in the case of mRNA, since the protein can be expressed in the cytoplasm even if the mRNA does not move into the nucleus, it is suitable for expressing the protein in non-dividing cells. In this case, protein expression is temporary, but such temporary expression is effective for many diseases or in cancer vaccine therapy (Non-patent Document 3). , 4).

R.W.Malone, P.L.Flenger, I.M.Verma, “Cationic Liposome-mediated RNA transfection.”, Proc. Natl. Acad. Sci. USA, 86 (1989), p.6077-6081 D. Lu, R. Benjamin, M. Kim, RM Conry, DT Curiel, “Optimization of methods to achieve mRNA-mediated transfection of tumor cells in vitro and in vivo exploying controlling ligand vectors.”, Cancer Gene Ther., 1 ( 1994), p.245-252 DM Anderson, LL Hall, AR Ayyalapu, VR Irion, MH Nantz and JG Hecker, “Stability of mRNA / Cationic Lipid Lipoplexes in Human and Rat Cerebrospinal fluid: Methids and Evidence for Nonviral mRNA Gene Delivery to the Central Nervous System.”, Human Gene Ther., 14 (2003), p.191-202 D.A. Mitchell, S.K.Nair, “RNA transfected dendritic cells as cancer vaccines.”, Curr. Opin. Mol. Ther., 2 (2000), p.176-181

  As described above, the substance introduction method using liposomes has improved the introduction efficiency by using cationic liposomes, but it is still not sufficient, and a method for introducing substances into cells more efficiently is desired. It was rare.

  The present invention has been proposed in view of such a conventional situation, and a liposome-based substance introduction carrier, substance introduction method, and substance introduction kit capable of introducing a substance into cells with high efficiency are provided. The purpose is to provide.

  In order to achieve the above-described object, the present inventors have conducted intensive research from various viewpoints. As a result, it was found that a substance can be introduced into cells with high efficiency by using a liposome combined with apatite particles as a substance introduction carrier. The present invention has been completed based on such findings.

  That is, the substance introduction carrier according to the present invention is characterized in that apatite particles are bound to liposomes.

  In addition, the substance introduction method according to the present invention forms a complex with a desired substance to be introduced into a cell, or incubates a substance introduction carrier encapsulating the desired substance with the target cell, thereby It is characterized in that a desired substance is introduced into.

  In addition, the substance introduction kit according to the present invention is characterized by including a calcium salt, bicarbonate or inorganic phosphate, and a liposome material.

  As described above, there has never been reported so far that a substance can be introduced into cells with high efficiency by using liposomes to which apatite particles are bound. It is.

  According to the present invention, a substance can be introduced into cells with high efficiency by using a liposome combined with apatite particles as a substance introduction carrier.

  Hereinafter, embodiments to which the present invention is applied will be described in detail with reference to specific experimental results.

Cell culture First, cells used in the experiment were cultured. Specifically, a DMEM (Dulbecco's modified Eagle's medium) medium containing 10% FBS (fetal bovine serum), 50 μg penicillin and streptomycin per ml of medium, and 100 μg neomycin per ml of medium in a 25 cm ² flask ( Gibco BRL) was added, and human cervical cancer cell line HeLa and mouse fibroblast cell line NIH3T3 were cultured in this medium under conditions of 37 ° C. and 5% CO ₂ .

  In addition, primary hepatocytes isolated from the liver of ICR mice (female, 5-7 weeks old) by modified in situ perfusion method were seeded in a 24-well plate coated with collagen, and similarly DMEM Cultured in medium. Since these primary hepatocytes are considered non-dividing cells, they are suitable for evaluating mRNA transfection.

Preparation of mRNA Next, mRNA was prepared as a desired substance to be introduced into cells. Specifically, using the SP6 RiboMAX ^TM kit (manufactured by Promega) according to the manual, in the presence or absence of m7G (5 ') pppG (5') cap analog (manufactured by Ambion), poly A tail ( A luciferase SP6 control DNA (Promega) having A30) was translated in vitro to obtain luciferase gene mRNA. The ratio of cap analog to GTP was 5: 1, and when no cap analog was used, the concentration of GTP was increased to 5 mM. When mRNA translated in vitro (including those with and without a cap structure) was confirmed by gel electrophoresis, it was about 1800 bp.

  Thereafter, in order to purify the mRNA, the mRNA was treated with RQ1 DNase1 (manufactured by Promega), the mRNA was extracted with phenol: chloroform: isoamyl alcohol and chloroform, and sodium acetate (pH 5.5) and ethanol were extracted. In addition, it was precipitated. The precipitated mRNA was washed with 70% ethanol and then dissolved in water. The obtained mRNA was quantified by measuring the absorbance at 260 nm, further denatured by heating at 65 ° C. for 15 minutes, and then confirmed by agarose gel electrophoresis.

Preparation of liposome Next, the liposome used for experiment was prepared. As a liposome material for forming liposomes, DOTAP (N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethyl ammonium chloride; manufactured by Sigma), which is a cationic lipid, was used. Specifically, chloroform was evaporated from DOTAP dispersed in chloroform, dispersed in distilled water, and then stirred for 10 minutes at a temperature (50 ° C.) higher than the gel-liquid crystal phase transition temperature of DOTAP. The obtained milky liquid was subjected to ultrasonic treatment for 10 minutes under temperature control using Branson sonifer 250 (Branson Sci., USA). Note that the ultrasonic power was minimized in order to prevent the milky liquid from foaming vigorously.

Preparation of mRNA-liposome-carbonate apatite particle complex Next, an mRNA-liposome-carbonate apatite particle complex used for introducing mRNA into cells was prepared. As controls, an mRNA-carbonate apatite particle complex and an mRNA-liposome complex were also produced.

Specifically, mRNA-carbonate apatite particle complex comprises 3 μg of heat-denatured mRNA and 4 μl of 1M CaCl ₂ in 1 ml of serum-free bicarbonate (HCO ₃ ⁻ ) buffer medium (DMEM medium or WE (Williams' E) It was prepared by adding to medium; pH 7.4), mixing, and incubating at 37 ° C. for 30 minutes.

  The mRNA-liposome complex was prepared by dissolving 3 μg of heat-denatured mRNA and 4-16 μg of DOTAP in 100 μl of DMEM medium (pH 7.5). After 15 minutes, DOTAP was added to the mRNA solution. It was prepared by incubating at room temperature for 40 minutes. Finally, the total amount was adjusted to 1 ml by adding DMEM medium (pH 7.5).

The mRNA-liposome-carbonate apatite particle complex is prepared by dissolving a heat-denatured mRNA and DOTAP in the same manner as described above in a bicarbonate buffered DMEM medium (pH 7.5) and incubating at room temperature for 15 minutes. Then, 4 mM CaCl ₂ was added to adjust the total volume to 1 ml, and the mixture was incubated at 37 ° C. for 30 minutes.

mRNA introduction into cells using mRNA-liposome-carbonate apatite particle complex and expression of luciferase in the cell Next, mRNA-liposome-carbonate apatite particle complex and mRNA-carbonate apatite particle complex as a control, Using the mRNA-liposome complex, mRNA was introduced into the cells, and the expression of luciferase in the cells was confirmed.

  Specifically, first, the day before the introduction of mRNA, cells synchronized with the growth process were seeded at a density of 100,000 cells / well in a 24-well plate and cultured.

  Subsequently, the washed cells were added with a solution containing each complex and FBS to a final concentration of 10%, and incubated at 37 ° C. for 2 to 4 hours. After 4 hours, the solution was replaced with fresh serum medium and incubated for an additional 6-12 hours. Thereafter, the cells were lysed in a lysis buffer (Promega), and luciferase activity was measured using a photon counter (TD-20 / 20 Luminometer, Promega). The luciferase activity was expressed as the amount of luminescence per mg of cell protein.

  FIG. 1 shows the expression status of luciferase when mRNA is introduced into 80% confluent HeLa cells. As can be seen from FIG. 1, at any concentration of DOTAP of 4 to 16 μg / ml, liposomes (DOTAP) can be obtained when mRNA is introduced using a liposome-carbonate apatite particle complex (DOTAP + CA). The expression efficiency of luciferase was about 10 times higher than when mRNA was introduced. From FIG. 1, since the optimal concentration of DOTAP was 6 to 10 μg / ml, the concentration of DOTAP was set to 6 to 10 μg / ml for NIH3T3 cells and primary hepatocytes.

  Subsequently, FIG. 2 shows the expression state of luciferase when mRNA is introduced into 80% confluent NIH3T3 cells. As can be seen from FIG. 2, the expression efficiency of luciferase was 9-14 times higher when the carbonate apatite particles were bound to the liposomes (+ CA) than when not bound (−CA).

  Subsequently, FIG. 3 shows the expression state of luciferase when mRNA is introduced into 50% confluent primary hepatocytes. As can be seen from FIG. 3, the expression efficiency of luciferase is approximately lower when mRNA is introduced using a liposome-carbonate apatite particle complex (DOTAP + CA) than when mRNA is introduced using a liposome (DOTAP). Ten times higher.

  Although not shown in FIGS. 1 to 3, when mRNA was introduced using carbonate apatite particles, the expression of luciferase was not confirmed.

Effect of the presence or absence of serum on luciferase expression efficiency Next, an mRNA-liposome-carbonate apatite particle complex using 8 μg / ml DOTAP and an mRNA-liposome complex as a control using 8 μg / ml DOTAP. The effect of the presence or absence of serum upon introduction of mRNA into the cell on the luciferase expression efficiency in the cell was confirmed.

  Specifically, first, the day before the introduction of mRNA, HeLa cells synchronized with the growth process were seeded at a density of 100,000 cells / well in a 24-well plate and cultured.

  Subsequently, a solution containing each complex with the washed cells, a sample added with FBS so as to have a final concentration of 10%, and a sample not added with FBS were prepared, and the sample was prepared at 37 ° C. for 2 to 4 hours. Incubated. After 4 hours, the solution was replaced with fresh serum medium and incubated for an additional 6-12 hours. Thereafter, the cells were lysed in a lysis buffer (Promega), and luciferase activity was measured using a photon counter (TD-20 / 20 Luminometer, Promega). The luciferase activity was expressed as the amount of luminescence per mg of cell protein.

  FIG. 4 shows the expression state of luciferase when mRNA is introduced into 80% confluent HeLa cells. As can be seen from FIG. 4, even when mRNA was introduced in the presence of serum (S +), the expression efficiency of luciferase decreased compared to the case where mRNA was introduced in the absence of serum (S−). It shows sufficient expression efficiency.

  As described above with reference to specific experimental results, mRNA can be introduced with high efficiency not only into dividing cells but also into non-dividing cells by using the mRNA-liposome-carbonate apatite particle complex. In particular, this mRNA introduction efficiency does not decrease even in the presence of serum. In addition, by applying this substance introduction method, a substance introduction kit containing calcium salt, bicarbonate, and liposome material can be realized.

  Here, as the first reason that the expression efficiency of mRNA is increased by binding carbonate apatite particles to liposomes, the concentration of the complex on the cell surface is increased by binding carbonate apatite particles to liposomes, As a result, it is assumed that the uptake of mRNA into the cell increased.

  In order to confirm this phenomenon, ethidium monoazide (EMA) -labeled mRNA was introduced into HeLa cells and observed with a fluorescence microscope 4 hours later. As a result, high levels of mRNA were incorporated into the cells. Furthermore, when the mRNA-liposome-carbonate apatite particle complex is used, the introduction efficiency reaches the maximum after 2 hours of incubation, whereas when the mRNA-liposome complex is used, 4 hours of incubation is required. Met. This also indicates that the uptake of the complex is promoted by the presence of the apatite particles.

  The second reason for the increased expression efficiency of mRNA is that carbonate apatite particles are pH-sensitive inorganic crystals and have the ability to neutralize endosomes. It is assumed that it is released into the cytoplasm.

  It should be noted that the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the scope of the present invention.

  For example, although carbonate apatite particles are used in the above-described embodiment, phosphate apatite particles may be used. In this case, a phosphate buffer medium may be used instead of the bicarbonate buffer medium, and the substance introduction kit may include inorganic phosphate instead of bicarbonate.

  Further, in the above-described embodiment, it has been described that a complex with mRNA is prepared and mRNA is introduced into a cell. However, a complex with a drug, a physiologically active substance, DNA, or the like is prepared, and these are added to a cell. You may make it introduce | transduce a substance.

  In the above-described embodiment, the complex is described. However, mRNA, a drug, a physiologically active substance, DNA, or the like may be encapsulated in a liposome combined with carbonate apatite particles. .

It is a figure which shows the expression condition of luciferase at the time of introduce | transducing mRNA with respect to a HeLa cell. It is a figure which shows the expression condition of luciferase at the time of introduce | transducing mRNA with respect to NIH3T3 cell. It is a figure which shows the expression condition of luciferase at the time of introduce | transducing mRNA with respect to a primary hepatocyte. It is a figure which shows the expression condition of luciferase at the time of introduce | transducing mRNA with respect to HeLa cell in presence of serum or absence.

Claims (6)

  A substance-introducing carrier characterized in that apatite particles are bound to liposomes.   2. The substance introduction carrier according to claim 1, wherein the liposome is a cationic liposome.   2. The substance-introduced carrier according to claim 1, wherein the apatite particles are carbonate apatite particles formed from bicarbonate ions and calcium ions.   The substance introduction carrier according to any one of claims 1 to 3, wherein the substance introduction carrier forms a complex with a desired substance to be introduced into a cell or is encapsulated with the desired substance.   A substance introduction method comprising introducing a desired substance into the target cell by incubating the substance introduction carrier according to claim 4 with the target cell.   A substance introduction kit comprising a calcium salt, a bicarbonate or an inorganic phosphate, and a liposome material.

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