JP4536655B2 - Cell introduction agent, cell introduction method, method for producing cell introduction agent, composition for producing cell introduction agent, and kit for producing cell introduction agent - Google Patents

Description

  The present invention relates to a cell introduction agent for introducing a target substance such as a polynucleotide into a cell, a cell introduction method, a method for producing a cell introduction agent, a composition for producing a cell introduction agent, and a kit for producing a cell introduction agent. About.

  Introduction of DNA into mammalian cells has become an extremely effective research technique for gene structure, function and control, and is expected in the fields of gene therapy and DNA vaccines. As a conventional gene transfer method, a virus method using a recombinant such as retrovirus or adenovirus as a vector for gene therapy is common.

  However, several problems have been pointed out in using the virus itself. That is, there is a risk of toxicity due to the appearance of an unexpected mutant virus, there is a possibility that the activity may be neutralized in vivo by the immunogenicity of the virus, and there is a restriction on the size of the gene that can be used. It has been pointed out that it can only be used in sizes, is difficult to manufacture and transport, and is expensive.

  For this reason, development of gene transfer (transfection) technology that does not use a virus instead of a virus vector has been actively carried out for basic research and application to an encounter therapy. Various non-viral gene transfer methods are known, such as the calcium phosphate method using a coprecipitate of DNA and calcium, and the lipofection method for forming complex particles of cationic lipids such as liposomes and anionic DNA. ing.

  These non-viral gene introduction methods are currently being actively developed for basic research and gene therapy applications. However, non-viral gene transfer technology is still inferior in gene transfer and expression efficiency compared to the virus method.

  The present invention has been made in view of the above, and is a cell introduction agent for introducing a target substance (drug, protein, polynucleotide, etc.) into a cell, and has high introduction efficiency into the cell. An object of the present invention is to provide a cell introduction agent, a cell introduction method, a cell introduction agent production method, a cell introduction agent production composition, and a cell introduction agent production kit having excellent reproducibility and biocompatibility. .

  As a result of diligent research to solve the above-mentioned problems, the present inventors have found that the complex particles of calcium phosphate-based material and DNA existed at pH 8.0 within a predetermined time after changing to pH 6.0. It was found that by adjusting the dissolution rate of the composite particles with respect to the pH so that at least 50% of the particles are dissolved, the release time of DNA in the cells can be controlled and efficient intracellular introduction can be realized. The present invention has been completed based on this finding.

  Complex particles are taken up into cells by endocytosis and released from endosomes into the cytoplasm. Since the pH in the endosome is acidic (about pH 5), the complex particles taken up by endocytosis are exposed to external pH changes from about pH 7 to pH 5. In the present invention, by adjusting the dissolution rate of the composite particle with respect to the pH within a predetermined range, the composite particle is rapidly dissolved after being taken into the cell to release DNA, so that the cell introduction efficiency is extremely high. It is realized.

  Conventionally, regarding the transfection efficiency in the calcium phosphate method, from the viewpoint of how efficiently complex particles are taken into cells and how efficiently they can escape from endosomes after being taken into cells. Consideration has been made. However, no example has been reported that considers the mechanism when complex particles dissolve and release DNA into cells after being taken up into cells. That is, the present invention is characterized in that the dissolution rate of the composite particles with respect to the pH is controlled so that the composite particles are rapidly dissolved using the acidic pH in the endosome after being taken into the cells.

That is, the present invention is as follows.
[1] A cell introduction agent for introducing a target substance into cells,
Containing composite particles composed of at least the target substance and a calcium phosphate-based material,
When changing from pH 8.0 to pH 6.0, at least 50% of the composite particles existing at pH 8.0 are dissolved within a predetermined time after changing to pH 6.0. A cell introduction agent characterized by the above.
[2] The cell introduction agent according to [1], wherein an average particle size of the composite particles is 500 nm or less.
[3] The cell introduction agent according to [1] or [2], wherein the calcium phosphate material is carbonate apatite.
[4] The cell introduction agent according to any one of [1] to [3], wherein the target substance is a negatively charged substance.
[5] The cell introduction according to any one of [1] to [4], wherein the target substance is at least one substance selected from the group consisting of drugs, proteins, and polynucleotides. Agent.
[6] A cell introduction method comprising introducing a target substance into a cell using the cell introduction agent according to any one of [1] to [5].
[7] A method for producing a cell introduction agent, which is used for introducing a target substance into cells and contains composite particles composed of the target substance and carbonate apatite,
A method for producing a cell introduction agent, comprising a step of forming the composite particles by preparing a composition containing at least calcium ions, phosphate ions, hydrogen carbonate ions, and the target substance.
[8] A step of preparing a first solution containing the calcium ions and the target substance,
Preparing a second solution containing the phosphate ions and the bicarbonate ions; and
The method for producing a cell introduction agent according to [7], comprising a step of preparing the composition by mixing the first solution and the second solution.
[9] The method for producing a cell introduction agent according to [7] or [8], wherein the calcium ion concentration of the composition is 0.1 mM or more.
[10] The method for producing a cell introduction agent according to any one of [7] to [9], wherein the phosphate ion concentration of the composition is 0.1 mM or more.
[11] The method for producing a cell introduction agent according to any one of [7] to [10], wherein the bicarbonate ion concentration of the composition is 1.0 mM or more.
[12] The method for producing a cell introduction agent according to any one of [7] to [11], wherein the composition further contains a fluorine ion or a strontium ion.
[13] The method for producing a cell introduction agent according to any one of [7] to [12], wherein the composition has a pH of 6.0 to 9.0.
[14] The method for producing a cell introduction agent according to any one of [7] to [13], wherein the composite particles are formed by maintaining the composition at 10 ° C or higher.
[15] A composition for producing a cell introduction agent for producing a cell introduction agent, which is used for introducing a target substance into cells and contains composite particles composed of the target substance and carbonate apatite. And
Contains at least calcium ions, phosphate ions, and bicarbonate ions,
A composition for producing a cell introduction agent, wherein the cell introduction agent is produced by adding the target substance.
[16] A composition for producing a cell introduction agent for producing a cell introduction agent, which is used for introducing a target substance into cells and contains composite particles composed of the target substance and carbonate apatite. And
Contains at least phosphate ions and bicarbonate ions,
A composition for producing a cell introduction agent, wherein the cell introduction agent is produced by adding the target substance and calcium ions.
[17] A kit for producing a cell introduction agent for producing a cell introduction agent, which is used for introducing a target substance into cells and contains composite particles composed of the target substance and carbonate apatite. ,
A first component containing at least calcium ions;
A second component containing at least phosphate ions and bicarbonate ions,
A cell introduction agent containing the composite particle is produced by adding the target substance to the first component and then mixing the first component and the second component. Production kit.

  By using the cell introduction agent of the present invention, the target substance can be introduced into cells extremely efficiently. The composite particles contained in the cell introduction agent of the present invention have a property that at least 50% is dissolved when pH 8.0 is changed to pH 6.0, so that the target substance is quickly released after being taken into the cells. To do. In addition, it is possible to introduce various substances into cells from low molecular weight drugs to polyanions having a relatively high molecular weight such as DNA. Moreover, since the composite particle contained in the cell introduction agent of the present invention is composed of a calcium phosphate-based material, it is easily taken into the cell and there is little concern about toxicity.

  In addition, by making the average particle size of the composite particles 500 nm or less, the efficiency of incorporation into cells can be further increased. Furthermore, by using carbonate apatite as the calcium phosphate material, growth into large crystals can be suppressed, and the efficiency of uptake into cells can be further increased. Further, by using a drug, protein, and polynucleotide as the target substance, it can be suitably used for disease treatment, gene recombination technology, RNA interference, and the like.

  In addition, since the cell introduction method of the present invention uses the cell introduction agent of the present invention, the target substance can be introduced into cells extremely efficiently. Therefore, it can be suitably used for disease treatment, gene recombination techniques, RNA interference, and the like.

  The method for producing a cell introduction agent of the present invention is composed of a target substance and carbonate apatite by preparing a composition containing at least calcium ions, phosphate ions, bicarbonate ions, and the target substance. This is a method for producing a cell introduction agent containing complex particles, and the cell introduction agent of the present invention can be produced easily. In the method for producing a cell introduction agent of the present invention, a first solution containing calcium ions and a target substance is prepared, and separately from this, a second solution containing phosphate ions and hydrogen carbonate ions is prepared. The production efficiency of the composite particles can be increased by preparing and mixing the first solution and the second solution to prepare the composition. Moreover, the production | generation efficiency of the said composite particle can be improved by making the calcium ion concentration of the said composition into 0.1 mM or more. Moreover, the production | generation efficiency of the said composite particle | grain can be improved by making the phosphate ion density | concentration of the said composition into 0.1 mM or more. Moreover, the production | generation efficiency of the said composite particle | grain can be improved by making the hydrogencarbonate ion density | concentration of the said composition into 1.0 mM or more. Moreover, the cell introduction agent with high cell introduction efficiency can be manufactured by controlling pH of the said composition to pH 6.0-9.0. Further, by maintaining the composition at 10 ° C. or higher, a cell introduction agent having high cell introduction efficiency can be produced.

  In addition, since the composition for producing a cell introduction agent of the present invention contains at least calcium ions, phosphate ions, and hydrogen carbonate ions, the cell introduction agent of the present invention can be simply added by adding the target substance. Can be easily prepared. Another composition for producing a cell introduction agent of the present invention contains at least a phosphate ion and a bicarbonate ion. Therefore, in use, the cell of the present invention can be obtained simply by adding the target substance and calcium ion. The introduction agent can be easily prepared.

  The kit for producing a cell introduction agent of the present invention comprises a first component containing at least calcium ions and a second component containing at least phosphate ions and hydrogen carbonate ions. A cell introduction agent capable of realizing high introduction efficiency can be prepared simply by adding a target substance to the mixture and mixing the first component and the second component.

Hereinafter, embodiments of the present invention will be described in detail in the following order.
[I] Cell transfer agent [II] Method for producing cell transfer agent [III] Cell transfer method

[I] Cell Introducing Agent The cell introducing agent of the present invention is used for introducing a target substance into cells, and contains at least composite particles composed of the target substance and a calcium phosphate-based material. It is.

  When the complex particles contained in the cell introduction agent of the present invention are changed from pH 8.0 to pH 6.0, the complex particles existed at pH 8.0 within a predetermined time after the change to pH 6.0. It is characterized in that at least 50% of the composite particles have the property of dissolving. Hereinafter, in the present specification, the property that the composite particles are dissolved by changing the pH from above pH 7 to below pH 7 is referred to as “pH solubility”.

  Since the pH in the endosome is acidic (about pH 5), the complex particles taken up by endocytosis are exposed to external pH changes from about pH 7 to pH 5, but the complex particles of the present invention are dissolved in pH. Because it is highly soluble, it dissolves quickly after being taken up into the cell and releases the target substance. Thereby, the target substance can be efficiently introduced into the cells.

  In the present invention, the pH solubility required for the composite particles is such that 50% or more of the composite particles existing at pH 8.0 are dissolved within a predetermined time by changing from pH 8.0 to pH 6.0. The level is sufficient, but in the present invention, the higher the pH solubility of the composite particles, the better.

  The high pH solubility of the composite particles includes (1) the range of pH change required for the composite particles to dissolve, (2) time required for the composite particles to dissolve, and (3) pH change. Is defined by three parameters of the proportion of composite particles dissolved. That is, the smaller the pH change required for the composite particles to dissolve, the higher the pH solubility of the composite particles. In addition, the shorter the time required for the composite particles to dissolve, the higher the pH solubility of the composite particles. Moreover, it means that the higher the ratio of the composite particles dissolved by pH change, the higher the pH solubility of the composite particles.

  In the present invention, the pH change required for 50% or more of the composite particles to dissolve is such that the pH value at the start is more than pH 7.0 and not more than 8.0 and the pH value after the pH change is pH 6. It is 0 or more and pH 7.0 or less. In this invention, it is so preferable that the range of this pH change is narrow. In the present invention, the pH value before pH change is more than pH 7.0 and pH 8.0 or less, preferably more than pH 7.0 and less than pH 7.5, more preferably more than pH 7.0 and less than pH 7.2, and still more preferably more than pH 7.0. It is preferable that the pH is 7.1 or less, particularly preferably more than pH 7.0 or less than pH 7.05, and most preferably more than pH 7.0 or less than pH 7.01. In addition, the pH value after pH change is pH 6.0 or more and pH 7.0 or less, preferably pH 6.5 or more and pH 7.0 or less, more preferably pH 6.7 or more and pH 7.0 or less, and further preferably pH 6.8 or more and pH 7. It is preferably 0 or less, more preferably pH 6.9 or more and pH 7.0 or less, particularly preferably pH 6.95 or more and pH 7.0 or less.

  The time required for 50% or more of the composite particles to dissolve is preferably within 10 minutes, more preferably within 5 minutes, further preferably within 2 minutes, and within 1 minute. It is particularly preferred that

  Further, the ratio of the composite particles dissolved by the pH change is preferably 50% or more, preferably 80% or more, more preferably 100% of the composite particles existing before the pH change.

  That is, it is preferable that the composite particles used in the present invention have a property of rapidly dissolving by changing the pH slightly to the acidic side.

  Such composite particles are composed of a target substance and a calcium phosphate material. The target substance can be used without particular limitation as long as it can form a composite particle with a calcium phosphate material. Specifically, drugs, polyanions such as proteins and polynucleotides can be used. Furthermore, the target substance in the present invention is preferably a negatively charged substance.

  Specific examples of drugs that can be used in the present invention include anticancer drugs and antitumor antibiotics. Specific examples of the anticancer agent include methotrexate (antifolate), vinblastine (Vinblastine, vinca alkaloid), anthracycline (Antracycline), daunomycin (Adriamysin), and the like. Antitumor antibiotics include Duocarmycin, Enedynes, Neocarzinostatin, Calicheamicin, Macrolide. By forming complex particles using such a drug, the intracellular introduction efficiency of the drug can be improved, and therefore, it can be suitably used for treating various diseases.

  As the polynucleotide, either DNA or RNA can be used, and a hybrid polynucleotide composed of DNA and RNA can also be used. For example, when gene recombination is performed using the cell introduction agent of the present invention, complex particles may be formed using vector DNA containing the gene to be expressed. Here, any DNA such as circular plasmid DNA, linear plasmid DNA, artificial chromosome, triplex DNA, etc. may be used as the DNA. Alternatively, complex particles may be formed using RNA capable of adjusting cell function, such as antisense RNA, siRNA that causes RNA interference.

In the present invention, the calcium phosphate material constituting the composite particle is a material mainly composed of Ca and PO ₄ . In the present invention, the calcium phosphate material is preferably apatites. As the apatites, hydroxyapatite, carbonate apatite and the like can be used, and it is particularly preferable to use carbonate apatite.

The carbonate apatite preferably used in the present invention is represented by Ca _10-m X _m (PO ₄ ) ₆ (CO ₃ ) _1-n Y _n . Here, X is an element that can partially replace Ca in carbonate apatite, and examples thereof include Sr, Mn, and rare earth elements. m is a positive number of 0 or more and 1 or less, preferably 0 or more and 0.1 or less, more preferably 0 or more and 0.01 or less, and particularly preferably 0 or more and 0.001 or less. . Y is a unit that can partially substitute CO ₃ in carbonate apatite, and examples thereof include OH, F, and Cl. n is a positive number of 0 or more and 0.1 or less, preferably 0 or more and 0.01 or less, more preferably 0 or more and 0.001 or less, and more preferably 0 or more and 0.0001 or less. Particularly preferred.

  The average particle size of the composite particles contained in the cell introduction agent of the present invention is preferably 500 nm or less, more preferably 400 nm or less, further preferably 300 nm or less, and particularly preferably 200 nm or less. The smaller the average particle size of the composite particles, the more efficient the incorporation of the composite particles into the cells. The lower limit of the average particle size of the composite particles is not particularly limited, but is usually 20 nm or more.

  The cell introduction agent of the present invention contains the composite particle. The cell introduction agent of the present invention is not particularly limited in its dosage form as long as it can be introduced into cells without denaturing the target substance, and any dosage form such as powder, solid, solution, etc. I do not care.

[II] Method for Producing Cell Introducing Agent Next, the method for producing the cell introducing agent of the present invention will be described. In the method for producing a cell introduction agent of the present invention, the composite particle is prepared by preparing a composition containing at least four components of at least calcium ions, phosphate ions, bicarbonate ions, and a target substance as essential components. It is a method characterized by forming. This composition is preferably prepared as an aqueous solution.

  Here, the calcium ion concentration in the composition is preferably 0.1 mM or more, more preferably 0.5 mM or more, and further preferably 1 mM or more. The calcium ion concentration is preferably 1 M or less, more preferably 100 mM or less, and even more preferably 10 mM or less.

  The phosphate ion concentration is preferably 0.1 mM or more, more preferably 0.5 mM or more, and even more preferably 1 mM or more. The phosphate ion concentration is preferably 1 M or less, more preferably 100 mM or less, and even more preferably 10 mM or less.

  The bicarbonate ion concentration is preferably 1.0 mM or more, more preferably 5 mM or more, and even more preferably 10 mM or more. The bicarbonate ion concentration is preferably 10 M or less, more preferably 1 M or less, and even more preferably 100 mM or less.

  The supply form of these ions into the composition is not particularly limited, but it is preferable to add these calcium ion source, phosphate ion source, and bicarbonate ion source salt to the aqueous solution. The amount added is preferably controlled so that the calcium ion concentration, phosphate ion concentration, and bicarbonate ion concentration are within the above-mentioned ranges.

  In addition, the concentration of the target substance in the composition is usually preferably 1 μg / L to 1 mg / L, but may be in excess or lower than this range as long as the target substance can be introduced into the cells.

  The mixing order of the four essential components is not particularly limited, and the composition may be prepared in any mixing order. As an example of a preferable preparation method, a first solution containing calcium ions and a target substance is prepared. In addition, separately, a second solution containing phosphate ions and bicarbonate ions is prepared, and the first solution and the second solution are mixed to prepare a composition containing the essential four components. Is preferred. Here, it is not necessary for the first solution to contain the entire amount of calcium ions that will eventually be included in the composition. That is, a part of calcium ions that will eventually be included in the composition may be contained in the first solution, and the remaining calcium ions may be contained in the second solution.

  When the calcium ion concentration of the composition is set to be relatively high, a final addition of the target substance may result in a carbonate apatite precipitate before forming a complex with the target substance. By mixing the first solution containing the target substance and the second solution containing phosphate ions and bicarbonate ions, composite particles can be formed efficiently.

In addition, Ca or CO ₃ in the carbonate apatite may be partially substituted by adding fluorine ions, chlorine ions, Sr, Mn or the like to the composition. Here, the addition amount of fluorine ions, chlorine ions, Sr, and Mn is set within a range that does not significantly affect the pH solubility and particle size range of the formed composite particles.

  In addition, other components can be added to the composition within a range not departing from the object of the present invention to form composite particles.

  For example, the composition may be prepared using a medium for culturing cells that are targets for introducing the target substance. Usually, when the cell introduction agent is added to the medium, the composition of the medium changes, and when the composition change is large, the concentration of each component contained in the cell introduction agent is calculated in advance, and the medium composition is finally obtained as intended. Therefore, the experimental operation becomes complicated. However, by preparing a composition containing the above-mentioned essential four components using a liquid medium, forming complex particles in this liquid medium, and using this as a cell introduction agent, by adding a cell introduction agent A significant change in the composition ratio of the components of the medium can be prevented.

  When preparing the composition using a medium, first, the medium is prepared as a liquid medium. And among the essential 4 components of calcium ion, phosphate ion, hydrogen carbonate ion, and the target substance, for the component not included in the liquid medium or the component that has not reached the required concentration, the corresponding component is added to the liquid medium. By supplementing, a composition containing the four essential components is prepared.

  Moreover, in order to prepare the said composition simply, the composition for cell introduction agent manufacture which can prepare the said composition only by adding the substance which is going to introduce | transduce into a cell can also be provided. Such a composition for producing a cell introduction agent is provided as a composition containing at least three components of calcium ion, phosphate ion and bicarbonate ion in a predetermined ratio. In use, if a predetermined amount of a target substance to be introduced into cells is added to the composition for producing a cell introduction agent, calcium ions, phosphate ions, hydrogen carbonate ions, and essential substances 4 of the target substance are required. Compositions containing the components can be easily prepared.

  It is also possible to provide a composition for producing a cell introduction agent capable of preparing the above composition simply by adding a target substance to be introduced into cells and calcium ions. Such a composition for producing a cell introduction agent is provided as a composition containing at least two components of phosphate ions and hydrogen carbonate ions in a predetermined ratio. In use, if a predetermined amount of the target substance and calcium ions are added to the composition for producing a cell introduction agent, the composition contains calcium ions, phosphate ions, hydrogen carbonate ions, and essential four components of the target substance. Can be easily prepared.

  Such a composition may be provided, for example, in the form of a solution, or in the form of a powder or a paste. In such a composition for producing a cell introduction agent, various ions may be contained in the form of a salt.

  Moreover, as another embodiment for easily preparing the composition, a cell comprising a first component containing at least calcium ions and a second component containing at least phosphate ions and hydrogen carbonate ions in a predetermined ratio. An introductory kit may be provided. When using the kit for producing a cell introduction agent, after mixing the target substance with the first component, the first component and the second component are mixed to obtain calcium ions, phosphate ions, bicarbonate ions, And the composition containing four essential components of the target substance can be prepared easily. Here, the first component and the second component may be provided in the form of a solution, or may be provided in the form of powder or paste. In the first component and the second component, various ions may be included in the form of a salt.

  After preparing a composition containing the essential four components of calcium ion, phosphate ion, hydrogen carbonate ion and the target substance, this composition is allowed to stand for a predetermined period of time to form a composite composed of the target substance and carbonate apatite. Body particles are formed.

  Here, it is preferable to form composite particles while maintaining the temperature of the composition at 10 ° C. or higher. More preferably, the temperature is maintained at 25 ° C. or higher, more preferably 37 ° C. or higher, particularly preferably 50 ° C. or higher. The upper limit of the temperature is not particularly limited as long as the target substance is not denatured, but there is no problem if it is usually 80 ° C. or lower, preferably 70 ° C. or lower.

  Moreover, it is preferable to adjust the pH of the composition to pH 6.0 to 9.0 to form composite particles. More preferably pH 7.0 or higher, further preferably pH 7.1 or higher, particularly preferably pH 7.2 or higher, most preferably pH 7.5 or higher, pH 9.0 or lower, further preferably pH 8.5. Hereinafter, the pH is particularly preferably 8.0 or less.

  After preparing the composition, the composition is allowed to stand until composite particles are formed. The time required for forming the composite particles is usually about 1 minute to 24 hours, and in many cases, composite particles that can be observed under a microscope are formed in about 10 minutes to 1 hour.

  The composition containing the composite particles can be used as a cell introduction agent as it is, but may be used after adding other components as necessary. Alternatively, the composite particles may be isolated from the composition to form a powder, or the composite particles may be solidified into a tablet or the like and used as a cell introduction agent.

[III] Cell Introduction Method The cell introduction method of the present invention is characterized by introducing a target substance into cells using the cell introduction agent of the present invention. Here, the target substance to be introduced into the cell is preferably a negatively charged substance that can adjust the cell function.

  In the present invention, various cells such as bacterial cells, actinomycetes cells, yeast cells, mold cells, plant cells, insect cells, and animal cells can be used as the target cells into which the target substance is introduced. Of these, animal cells, particularly mammalian cells, can be preferably used. The target cells into which the target substance is introduced include both in vitro and in vivo. That is, any cell such as a cultured cell, a cultured tissue, or a living body may be used.

  In the case of using cultured cells, a target substance can be introduced into the cells by preparing a medium containing the cell introduction agent of the present invention and culturing under normal culture conditions using the medium.

  Further, when the cell introduction agent of the present invention is used as a medicament for treating various diseases, for example, a cell introduction agent containing complex particles composed of a substance having a pharmacological activity and a calcium phosphate material is prepared and used. Substances having pharmacological activity can also be directly introduced into living cells by administration into the subcutaneous, intramuscular, intraperitoneal, or intravascular space of mammals (including humans).

  In addition, when used as a drug for gene therapy, introduction of a cell containing complex particles composed of a polynucleotide (eg, vector DNA, antisense RNA, RNAi, etc.) capable of adjusting cell function and a calcium phosphate material. An agent can be prepared and introduced into target cells and expressed. Examples of the disease for which gene therapy is performed include diseases such as cancer and genetic diseases.

  Hereinafter, the present invention will be described in more detail based on examples. In addition, this invention is not limited to the following Example.

The reagents used in the examples are as follows. PGL3 (Promega) containing luciferase under SV40 promoter control and pEGFP-N2 (Clontech) containing GFP gene under CMV control are XL-1 Blue (by the method shown in Molecular Cloning). And purified by Qiagen plasmid kit. Propidium iodide (PI) and LysoSensor ^™ Green DND-189 were purchased from Sigma and Molecular Probe, respectively. Lipofectamine and DMEM (catalog number 12800) were purchased from Gibco BRL.

As for cell culture, the HeLa, HepG2 and NIH3T3 cell lines were 10% fetal bovine serum (FBS), 50 μg / ml penicillin, 50 μg / ml streptomycin, 100 μg / ml neomycin-containing Dulbecco's modified Eagle's in 75 cm ² flasks. The cells were cultured in medium (DMEM, Gibco BRL) at 37 ° C. in a 5% CO ₂ atmosphere. Primary cultured hepatocytes were isolated from the livers of male ICR mice (5-7 weeks old) (SLC, Shizuoka, Japan) using a known modified in situ perfusion method and coated with collagen 24 It seed | inoculated to the well plate and it culture | cultivated using Williams'E (WE) culture medium (Gibco BRL) similarly instead of DMEM.

Example 1 Cell Introducing Agent [1] Preparation of Cell Introducing Agent 3-6 μl of 1M calcium chloride solution was suspended in 1 ml of serum-free bicarbonate buffer (DMEM or WE) medium (pH 7.5) containing 2 μl of plasmid DNA. The mixture was cultured at 37 ° C. for 30 minutes to produce DNA / carbonate apatite composite particles.

The composition of the used DMEM medium and WE medium is shown below.

  Fluorine-containing or strontium-containing carbonate apatite was produced by adding 0.01 to 3 μl of 1M sodium fluoride or strontium chloride together with calcium ions and DNA and culturing under the same conditions as above.

  By adding only 3 mM calcium ion to a cell culture medium (DMEM or Williams'E medium, pH 7.5) in which bicarbonate ions have a buffering effect, and culturing at 37 ° C. for 30 minutes under a microscope Particles that can be observed were produced. This particle formation occurs in a medium or solution buffered with bicarbonate ions, and a Hepes buffer medium or solution (pH 7.5) containing the same amount of total calcium ions (4.8 mM) and total phosphate (0.9 mM). ) Did not occur. This suggests that carbonate, together with phosphate and calcium ions, is involved in particle formation.

[2] Analysis of generated particles The generated particles were analyzed by chemical analysis, infrared spectroscopy, and X-ray diffraction.

(Chemical analysis)
In the same manner as above, carbonate apatite was produced using 6 mM calcium ions without adding DNA, and the particles precipitated by centrifugation and distilled deionized water were lyophilized. Other apatite particles were purified as described above and lyophilized. Calcium and phosphorus components were measured using an SPS 1500 VR Atomic Absorption Spectrophotometer. Carbon and fluorine were measured using CHNS-932 (Leco, USA) and SX-elements microanalyzer, YS-10 (Yanaco, Japan), respectively.

(Infrared spectroscopy)
The Fourier transform infrared spectroscopic analysis of the apatite particles prepared as described above was performed using FT / IR-230 (JASCO). Samples were placed in a mortar and about 1 mg was thoroughly mixed with 300 mg of spectroscopic potassium bromide. Thin pellets were prepared by applying a weight of 8000 kg in a potassium bromide pane under 0.5 torr depressurization.

(X-ray diffraction)
X-ray powder analysis of the prepared particles was performed using an M18XHF-SRA diffraction system.

Elemental analysis revealed 3% carbon, 17% phosphorus, and 32% calcium ions. From the peaks between 1410 and 1540 cm ⁻¹ and about 880 cm ⁻¹ of the FT-IR spectrum shown in FIG. 1-1, it is confirmed that the produced particles contain carbonate, and that the generated particles show 1000 to 1100 cm ⁻ shown in FIG. ^From the peaks at ¹ and 550 to 650 cm ⁻¹ , it was found that phosphoric acid was further contained. The X-ray diffraction pattern (FIGS. 1-3) shows that carbonate apatite is less crystalline than hydroxyapatite (FIGS. 1-4), as symbolized by its broad diffraction peak. This is a natural property of carbonate apatite.

Example 2 Gene transfer into cells [1] Expression level measurement of luciferase To examine the transfection efficiency of the cell transfer agent of the present invention prepared in Example 1 [1], luciferase using the cell transfer agent of the present invention was examined. The expression level was measured.

  About 50000 cells in a logarithmic growth phase per well of a 24-well plate were seeded the day before gene transfer. The prepared medium containing the DNA-containing particles was added to the cells washed with 10% FBS.

  After culturing for 4 hours, the medium was replaced with a medium containing serum, and the cells were cultured for 1 day. Luciferase gene expression was measured using a commercially available kit (Promega) and photon counting (TD-20 / 20 luminometer, USA). Each gene transfer experiment was repeated three times, and transfection efficiency was expressed as the average value of light intensity divided by the amount of protein (mg) in the cells.

Primary hepatocytes were seeded in a 24-well plate coated with collagen, and transformed after 3 hours of culture. Transformation by coprecipitation of calcium phosphate-DNA is described in Jordan M. et al. (Jordan, M., Schallhorn, A. & Wurm FM. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic acids research 24, 596-601 (1996)). Briefly, 12 μg of plasmid DNA was added in 300 μl of 250 mM calcium chloride solution. This solution is added to 300 μl of 2 × HBS (50 mM Hepes, 140 mM sodium chloride, 1.5 mM Na ₂ HPO ₄ .2H ₂ 0, pH 7.05) and immediately stirred by gently pipetting twice. did.

  The DNA / calcium phosphate mixture was left at room temperature for each indicated time. 100 μl of the mixed solution that had been left at room temperature in 1 ml of serum-containing medium was dropped, and cultured in that medium for 4 hours. After replacing with fresh serum-containing medium as described above, the culture was continued for one day.

  In order to investigate the effects of calcium ion concentration, pH of bicarbonate ion buffer medium, and culture temperature, transfection efficiency was improved by gene introduction using HeLa cells in medium containing 10% FBS by changing the calcium ion concentration and culture temperature. evaluated.

  Fig. 2-1 shows the transfection efficiency when the calcium ion concentration added in DMEM in the production of DNA / carbonate apatite particles depending on the pH of the bicarbonate ion buffer medium is changed. Fig. 6 shows transfection efficiency when the temperature in the production of DNA / carbonate apatite particles depending on the pH of a hydrogen ion buffer medium is changed. Here, transfection efficiency is examined by the expression level of luciferase in gene transfer using HeLa cells in a medium containing 10% FBS.

  The production of carbonate apatite particles was dependent on the calcium ion concentration, the pH of the carbonate ion buffer medium, and the culture temperature. The best calcium ion concentration needed to produce a sufficient amount of DNA / carbonated apatite complex to obtain high transfection efficiency is determined by the pH (Figure 2-1) or temperature (Figure 2-2) of the medium. It had a negative correlation. The decrease in efficiency compared to the initial high-efficiency transfection is attributed to the reduced amount of particles formed (observation results with a microscope). Improvements in the pH of the liquid medium, the culture temperature and / or the calcium ion concentration contribute to the formation of a supersaturated solution that is necessary for the production of particles. We were able to develop a flexible transfection system for the first time that high reproducibility can be achieved simply by controlling all parameters related to particle formation.

  In order to evaluate the role of carbonate apatite as an effective gene carrier, the present inventor has developed a variety of transfection methods, including two frequently used transfection methods, namely the calcium phosphate coprecipitation method and the lipofection method described above. We compared transfection efficiency.

  Fig.2-3 shows the gene expression level when HeLa cells are used to introduce a gene with carbonate apatite, hydroxyapatite, and lipofectamine, and Fig.2-4 shows that the HepG2 cells are used with carbonate apatite, hydroxya Figure 2-5 shows the gene expression level when gene transfer using apatite and lipofectamine is performed. Fig. 2-5 shows the gene expression level when gene transfer using carbonate apatite, hydroxyapatite and lipofectamine is performed using NIH3T3 cells. Fig. 2-6 shows the gene expression levels when gene introduction with carbonate apatite, hydroxyapatite, and lipofectamine was performed using mouse primary cultured hepatocytes.

  Here, the DNA / carbonate apatite complex particles are 3 to 6 mM calcium ion concentration (3 mM for all calcium ions not specified) and 2 μg plasmid DNA in 1 ml bicarbonate ion buffer (pH 7.5). And left at 37 ° C. for 30 minutes. In addition, the hot water of the HeLa cell shown in FIG. 2-3 is obtained by adding 6 mM calcium ion together with 2 μg DNA to 1 ml of serum-free medium (shown above) to produce particles, and the medium is made 100 μ1 (200 ng DNA). Alternatively, 20 μl (40 ng DNA) was used for gene transfer experiments in a medium containing FBS. DNA / hydroxyapatite composite particles are described in Jordan M .; It was produced according to these methods. The DNA / lipofectamine complex was prepared according to the manufacturer's protocol at a weight ratio of 1: 6. The gene was introduced into the cells by the method described above, and 2 μg of DNA was added to each well of the tissue culture plate (unless otherwise stated), and the method was different.

  For example, in HeLa cells, the luciferase expression level when transfected using carbonate apatite was 15 times or more and 25 times or more higher when compared with lipofection and calcium phosphate coprecipitation (FIG. 2-3). Nanogram amounts of DNA were sufficient for efficient transgene expression (FIGS. 2-3).

  The results showed that the transfection efficiency was significantly higher in HepG2 (FIG. 2-4), NIH3T3 cells (FIG. 2-5), and mouse primary cultured hepatocytes (FIG. 2-6).

  As a result of treating cells with EDTA after 4 hours of DNA uptake into cells, the gene expression level of luciferase was reduced by half (Fig. 2-3). This suggests that it contributes but offsets the hydrolysis of DNA by nucleolytic enzymes, which is also seen by the persistence of the fluorescence intensity of the PI-labeled DNA (not shown).

[2] MTT assay In order to clarify that the transfection efficiency did not appear to be high due to the high cell viability, the MTT assay was performed using HeLa cells.

  HeLa cells were cultured for 1 day after the gene introduction by the above method. 30 μl of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) solution (5 mg / ml) was added into each well and incubated for 4 hours. 0.5 ml DMSO was added after removing the medium. After the crystals were dissolved and incubated at 37 ° C. for 5 minutes, absorbance at a wavelength of 570 nm was measured with a microplate reader using a wavelength of 630 nm as a reference wavelength.

  Fig. 2-7 shows the results of examination of cell viability in HeLa cells by MTT assay.

[3] Analysis of complex particles contained in cell introduction agent (scanning electron microscope (SEM))
Drops of the DNA-carbonate apatite mixture prepared as described in the gene transfer protocol were added to a carbon-coated SEM stage, dried, and then observed with SEM (S-800, Hitachi, Japan).

  Fig. 3-1 shows a scanning electronic microscopic image. The scale bar is 600 nm.

  In the presence of an apatite structure, carbonate limits the growth of apatite crystals and increases solubility. The present inventors observed the produced carbonate apatite with a scanning electron microscope, and clarified that the crystal growth was mostly suppressed from a diameter of 50 nm to 300 nm (see FIG. 3-1, property submission). (Refer to the figure submitted.)

(Confocal laser microscope)
Since large particles are less likely to be taken in by phagocytosis than small particles, the present inventors have verified the effect of suppressing the size of carbonate crystals by using a PI (propidium iodide) labeled plasmid adsorbed on apatite. This was done by observing DNA uptake into cells.

  The pGL3 vector was labeled with PI so that the PI / DNA ratio was 1: 1, and particles generated with this labeled plasmid (generated according to the gene transfer protocol) were incubated with HeLa cells for 6 hours. Acidic sites were labeled using a 5 μM Lyso Sensor according to the method indicated by Molecular Probe. And after removing the particle | grains couple | bonded with the cell membrane using 5 mM EDTA melt | dissolved in PBS, it observed using LEICA TCS-NT.

  Fig. 3-2 (see the figure submitted in the property submission form) shows the release and expression of DNA via apatite. In FIG. 3-2, no DNA uptake is observed in (a) (control experiment). This is because endocytosis is blocked by energy consumption (addition of 50 mM 2-deoxyglucose and 1 mM sodium azide). DNA / carbonate apatite particles were generated using 6 mM calcium ions and 2 μg DNA in 1 ml serum-free medium (as described with reference to FIG. 2). There were 40 ng (b) and 200 ng (c) of DNA in 20 μl and 100 μl of a 1 ml suspension, respectively, and the cells were taken up for 4 hours. In (d), 2 μg of DNA was adsorbed on hydroxyapatite and taken up into cells for the same time.

  When particles are formed by culturing for 1 minute, using carbonate apatite (FIG. 3-2 (c)) is at least 10 times more efficient than using hydroxyapatite (FIG. 3-2 (d)). DNA was taken up into the cells.

  Incubating for a longer time (30 minutes) to form particles produces huge hydroxyapatite particles, and the efficiency of DNA incorporation into cells is significantly reduced (not shown in the text). Remarkably decreased (FIGS. 2-3).

  Therefore, the present inventors have found that carbonate apatite makes it easier to incorporate DNA into cells due to its inherent property of suppressing crystal growth and is a better gene transfer carrier than hydroxyapatite. Shows.

  To assess the role of DNA escape from endosomes in transgene expression, following endocytosis of PI-labeled plasmid DNA, we labeled endosomes with LysoSensor (endosomal fluorescent probe). As a result, 6 hours after the uptake of DNA by the cells, it was found that a lot of DNA was released from the endosome after the two co-localized (Figure 3-3, Figure submitted in the property submission form). See).

[4] Effect of pH in cell introduction In order to investigate the effect of pH in cell introduction, transfection was performed using bafilomycin Al, a specific inhibitor of V-ATPase (a proton pump used to acidify endosomes). The change in the injection efficiency was investigated. HeLa cells were cultured with DNA / carbonate apatite complex particles and 200 nM bafilomycin Al for 6 hours. After washing with PBS containing 5 mM EDTA, the cells were cultured for 1 day, and the expression of luciferase was detected. The results are shown in Fig. 4-1.

  Treatment with bafilomycin Al, a specific inhibitor of V-ATPase (a proton pump used to acidify endosomes), dramatically reduced the transfection efficiency of HeLa cells. This result indicates that an acidic environment is required to dissolve carbonate apatite and allow DNA to escape from the apatite.

  To examine this hypothesis, we added fluoride to reduce the solubility of apatite, so we synthesized carbonate apatite to which fluorine was added, and examined the effect of particle solubility on transfection efficiency. Changes in luciferase expression when the concentration of fluorine ions (0.01 to 3 mM) or strontium ions (0.01 to 3 mM) added when forming DNA / carbonate apatite particles was increased were examined. The results are shown in Fig. 4-2.

  Surprisingly, increasing the amount of fluorine contained in carbonate apatite significantly (100 times) reduced transfection efficiency (FIG. 4-2).

  In order to show that the transfection efficiency of fluorinated carbonate apatite was not due to a decrease in DNA uptake into cells, the present inventors introduced a pEGFP plasmid labeled with PI.

  Fig. 4-3 (see the figure submitted in the property submission form) shows the results of observation of uptake of PI-labeled plasmid DNA (pEGFP-N2) and expression of GFP protein by carbonate apatite and fluorinated carbonate apatite. . After culturing the particles containing DNA for 4 hours, the cells were further cultured for 20 hours, treated with 5 mM EDTA in PBS, and then observed.

  Although the amount of plasmid DNA incorporated into the cells was the same, the expression of GFP was observed in almost 50% of the cells with carbonate apatite (FIG. 4-3, upper panel), whereas carbon dioxide added with fluorine. When apatite was used, cells expressing GFP were not seen (FIG. 4-3, lower row).

  To promote the efficiency of transgene expression when the DNA uptake period is short, we investigated the effect of slightly reducing the solubility of carbonate apatite by adding a small amount of fluorine.

  FIG. 4-4 shows changes in the expression level of luciferase when adding μM level fluorine ions during the formation of DNA / carbonate apatite particles. Here, after culturing the cells with the particles, the cells were washed with EDTA and further cultured as described above for one day.

  Surprisingly, the addition of 1 μM sodium fluoride during the production of carbonate apatite promoted luciferase gene expression 15-fold. Further, when the pH of the endosome was raised and treated with chloroquine, which is known to protect DNA against nucleolytic enzymes, similar gene expression was observed (FIGS. 4-4).

  From these results, while the endosome buffering action and proton consumption are caused by crystals containing a small amount of fluorine ions, DNA is released at an intermediate rate, so that DNA degradation by remarkable nucleolytic enzymes is prevented. This shows that the fluorescence of PI is sustained (not shown in the figure), indicating that more complete DNA is present during transcription and translation.

[5] Study on pH-dependent solubility of apatite In order to investigate the relationship between apatite solubility and transfection efficiency, the turbidity of the solution after addition of acid was measured as an index of solubility. did.

  The solubility characteristics of the different apatites, generated according to the transfection protocol using 6 mM calcium ion solution without DNA, were adjusted from 7.0 to 6.8 with 1 M HCl and V-500 (Jasco , Japan) Using a spectrophotometer, turbidity at 320 nm was measured at different intervals and examined. The SPS 1500 VR atomic absorption spectrophotometer is an acetic acid buffer solution (pH 6.5 solution and pH 5.5 solution) at 37 ° C. after gently shaking the freeze-dried different apatite powder (10 rpm for 6 hours). Used to detect calcium ions released in it.

  Fluorine-added carbonate apatite prepared by adding 0 to 3 mM fluoride ions when producing carbonate apatite at pH 7.5 in Fig. 5-1 (see the figure submitted in the property submission form) (described in the experimental protocol) ) Is measured by turbidity at a wavelength of 320 nm immediately after adjusting the pH to 7.0 by adding 1N hydrochloric acid to the apatite suspension.

  After adjusting the pH from 7.5 to 7.0 by adding 1N hydrochloric acid, the solubility of carbonate apatite produced by increasing the concentration of sodium fluoride to be added gradually increases as shown by the change in turbidity. It decreased (FIG. 5-1). This result was consistent with the result that the transfection efficiency of carbonate apatite to which fluorine was added gradually decreased (FIG. 4-2).

  In order to examine whether the solubility of apatite is related to the degree of crystallization, we examined X-ray scattering of apatite (FIGS. 1-4, 1-5, 1-6). As a result, it was found that the higher the crystallinity, the lower the solubility (FIG. 5-1). In other words, apatite with high crystallinity (FIGS. 1-4 and 1-8) shows low transfection efficiency (FIGS. 2-3 and 4-2).

  Transfection efficiency decreases later (FIG. 4-2) due to the increase in crystallinity (FIGS. 1-6 to 1-8) accompanying the increase in fluorine content in carbonate apatite. Although we show that the decrease in transfection efficiency is due to the solubility of carbonate apatite with the addition of fluorine and not the effect of adding other fluorine, we have the effect of strontium when added to carbonate apatite. Was examined. Strontium increases the crystallinity of apatite and is less effective than fluorine, but decreases solubility.

  The solubility of carbonate apatite containing carbonate apatite, fluorinated carbonate apatite, and strontium at pH 7.0 and 6.8 is shown in FIG.

  By reducing the pH from 7.5 to 7.0 and 6.8, the carbonate apatite was completely dissolved within 1 minute, whereas the carbonate apatite with the addition of fluorine was only partially dissolved. (FIG. 5-2).

  As expected, when strontium chloride was added during the precipitation of carbonate apatite, the solubility decreased not as much as when fluorine was added (FIG. 5-2). Furthermore, the transfection efficiency was gradually decreased by increasing the concentration of strontium chloride during the production of DNA / carbonate apatite composite particles (FIG. 4-2). From these results, our findings clearly suggest that the release of DNA in the cytoplasm by lysis of apatite plays an important role in transfection via carbonate apatite.

  The present inventors compared the solubility of apatite containing hydroxyapatite by atomic emission spectrometry measurement of calcium ions released from the apatite powder with sodium acetate buffers at pH 6.5 and 5.5. The solubility of fluorinated carbonate apatite was lower than that of carbonate apatite and higher than that of hydroxyapatite (see FIG. 5-3).

  In order to demonstrate that the release of DNA associated with crystals in endosomes is a truly important factor for gene expression, we have used PI (pH sensitive dye) labeled plasmid DNA for 4 hours. After intracellular uptake, DNA bound to the cell surface was removed by EDTA (see FIGS. 5-4-a and d, see the figure submitted in the property submission form, the same applies below) and left for another 5 hours (FIG. 5). -4-b, c, e). It can be seen that the fluorescence intensity of PI is significantly reduced in free plasmid DNA after 5 hours due to the action of nucleolytic enzyme (FIGS. 5-4-b and e). Moreover, it turns out that the fluorescence intensity is hold | maintained in the cell processed with bafilomycin A1 after intracellular uptake | capture for 4 hours (FIG. 5-4-c).

  Although the fluorescence intensity of DNA (Fig. 5-4-f) taken into cells by the fluorinated carbonate apatite is hardly decreased (Fig. 5-4-g), the hydrolysis rate is high. In other words, DNA release is essential for gene expression. Furthermore, since the accumulation of a large amount of protons induced by V-ATPase due to the dissolution of crystals leads to the inflow of inactivated chlorine into the endosome, leading to the expansion and rupture of endosomes, the high solubility of carbonate apatite makes the cytoplasm of DNA It seems to contribute to the destabilization of endosomes during release into the body (Fig. 3-3).

  As described above, since the cell introduction agent according to the present invention has high cell introduction efficiency and excellent reproducibility and biocompatibility, it can be suitably used for gene recombination techniques, treatment of various diseases, and the like. .

It is a figure which shows IR spectrum of the produced | generated carbonate apatite. It is a figure which shows IR spectrum of the produced | generated hydroxyapatite. It is a figure which shows IR spectrum of the hydroxyapatite produced | generated by incubating in a bicarbonate ion buffer DMEM culture medium at 37 degreeC for 4 hours. It is a figure which shows the X-ray-diffraction pattern of a carbonate apatite. It is a figure which shows the X-ray-diffraction pattern of a hydroxyapatite. It is a figure which shows the X-ray-diffraction pattern of the fluorinated carbonate apatite containing 0.65% fluorine ion. It is a figure which shows the X repetition diffraction pattern of the fluorinated carbonate apatite containing 1.43% fluorine ion. It is a figure which shows the X-ray-diffraction pattern of the fluorinated carbonate apatite containing 2.5% fluorine ion. It is a figure which shows the transfection efficiency when changing the pH in the production | generation of DNA / carbonate apatite particle | grains and implementing gene transfer using a HeLa cell in a 10% FBS containing medium. It is a figure which shows the transfection efficiency when the temperature in the production | generation of DNA / carbonate apatite particle | grains is changed and the gene transfer using a HeLa cell is implemented in the culture medium containing 10% FBS. It is a figure which shows a gene expression level when the gene introduction | transduction by a carbonate apatite, a hydroxyapatite, and lipofectamine is performed using a HeLa cell. It is a figure which shows a gene expression level when the gene introduction | transduction by carbonate apatite, a hydroxyapatite, and lipofectamine is performed using HepG2 cell. It is a figure which shows the gene expression level when the gene introduction | transduction by carbonate apatite, a hydroxyapatite, and lipofectamine is performed using NIH3T3 cell. It is a figure which shows the gene expression level when the gene introduction | transduction by carbonate apatite, a hydroxyapatite, and lipofectamine is performed using the mouse | mouth primary culture hepatocytes. It is a figure which shows the result of having examined the cell viability in the HeLa cell by MTT assay. It is a figure which shows the scanning electron micrograph (scale bar is 600 nm) of the uptake | capture of DNA in the cell through a carbonate apatite. It is a figure which shows the uptake | capture into the cell of the PI-labeled plasmid DNA couple | bonded with the carbonate apatite and the hydroxyapatite. (A) shows no DNA uptake (control experiment). Endocytosis is blocked by energy consumption (addition of 50 mM 2-deoxyglucose and 1 mM sodium azide). DNA / carbonate apatite particles were generated in 1 ml serum-free medium using 6 mM calcium ions and 2 μg DNA. There were 40 ng (b) and 200 ng (c) of DNA in 20 μl and 100 μl of a 1 ml particle suspension, respectively, which were incorporated into cells for 4 hours. In (d), 2 μg of DNA was adsorbed on hydroxyapatite and taken up into cells for the same time. FIG. 3 shows the escape from endosome of PI-labeled DNA taken up by endocytosis. It was clearly seen after co-localization with the endosomal fluorescent probe (LysoSensor). It is a figure which shows the influence of bafilomycin A1 (inhibitor of V-ATPase) in cell introduction | transduction. HeLa cells were cultured with DNA / carbonate apatite complex particles and 200 nM bafilomycin A1 for 6 hours. After washing with PBS containing 5 mM EDTA, the cells were cultured for 1 day, and the expression of luciferase was detected. It is a figure which shows the change of a luciferase expression when raising the density | concentration of the fluorine ion (0.01 to 3 mM) or strontium (0.01 to 3 mM) added when forming DNA / carbonate apatite particle | grains. It is a figure which shows uptake | capture of PI-labeled plasmid DNA (pEGFP-N2) and expression of GFP protein by carbonate apatite (upper stage) and fluorine addition carbonate apatite (lower stage). The particles containing DNA were cultured for 4 hours, treated with PBS containing 5 mM EDTA, and then further cultured for 20 hours for observation. It is a figure which shows the change of the luciferase expression level when the fluoride ion of a micromol level is added at the time of formation of DNA / carbonate apatite particle. As described above, after culturing the cells with the particles, the cells were washed with EDTA and further cultured for 1 day. It is a figure which shows the dissolution behavior of apatite depending on pH. The solubility at pH 7.0 of fluorinated carbonate apatite prepared by adding 0 to 3 mM fluoride ions when producing carbonate apatite at pH 7.5 (as shown in the experimental method) Immediately after adjusting to pH 7.0 by adding hydrochloric acid, the turbidity at a wavelength of 320 nm was measured. It is a figure which shows the dissolution rate of the carbonate apatite containing a carbonate apatite, a fluorine addition carbonate apatite, and strontium when changing pH7.5 to pH7.0 and 6.8. It is a figure which shows the melt | dissolution rate to the acetic acid buffer of pH 6.5 and 5.5 of a carbonate apatite, a fluorine addition carbonate apatite, and a hydroxyapatite powder. The rate of calcium ion release from apatite was evaluated. FIG. 3 shows intracellular release of PI-labeled plasmid DNA from apatite crystals. Observation and measurement were performed according to the fluorescence intensity of PI. The cells were cultured with DNA / carbonate apatite for 4 hours (a, d), further treated with EDTA and then cultured for 5 hours (b, c, e), b and e were in the absence of bafilomycin A, c Is the condition in which 200 nM bafilomycin A is present. Similarly, when the cells were cultured with DNA / fluorinated carbonate apatite for 4 hours (f), and when they were further treated with EDTA and cultured for 5 hours (g). White and pink scales indicate 20 μm and 50 μm, respectively.

Claims (17)

A method for producing a cell introduction agent, which is used for introducing a polynucleotide into a cell and contains composite particles composed of the polynucleotide and carbonate apatite,
A method for producing a cell introduction agent, comprising a step of forming the composite particle by preparing a composition containing at least calcium ion, phosphate ion, bicarbonate ion and the polynucleotide . Preparing a first solution containing the calcium ions and the polynucleotide ;
Preparing a second solution containing the phosphate ions and the bicarbonate ions; and
The method for producing a cell introduction agent according to claim 1 , comprising a step of preparing the composition by mixing the first solution and the second solution. The method for producing a cell introduction agent according to claim 1 or 2 , wherein the composition has a calcium ion concentration of 0.1 mM or more. The method for producing a cell introduction agent according to any one of claims 1 to 3 , wherein the phosphate ion concentration of the composition is 0.1 mM or more. The method for producing a cell introduction agent according to any one of claims 1 to 4 , wherein the bicarbonate ion concentration of the composition is 1.0 mM or more. The method for producing a cell introduction agent according to any one of claims 1 to 5 , wherein the composition further contains a fluorine ion or a strontium ion. The pH of the composition, manufacturing method of the cell transfer agent according to any one of claims 1 to 6, characterized in that the pH 6.0 to 9.0. The method for producing a cell introduction agent according to any one of claims 1 to 7 , wherein the composite particles are formed by maintaining the composition at 10 ° C or higher. The cell introduction agent according to any one of claims 1 to 8, wherein the cell introduction agent is used for introducing a polynucleotide into a cell of an animal cell isolated from a human or non-human. Manufacturing method. The method for producing a cell introduction agent according to claim 1, wherein the composite particles have an average particle size of 500 nm or less. The method for producing a cell introduction agent according to any one of claims 1 to 11, wherein the polynucleotide is DNA and / or RNA . A polynucleotide is introduced into a cell isolated from a human or a non-human animal cell using the cell introduction agent produced by the production method according to any one of claims 1 to 11. A cell introduction method characterized by the above. The cell introduction method according to claim 12, wherein the polynucleotide is DNA and / or RNA . A composition for producing a cell introduction agent for producing a cell introduction agent, which is used for introducing a polynucleotide into a cell and contains composite particles composed of the polynucleotide and carbonate apatite,
Contains at least calcium ions, phosphate ions, and bicarbonate ions,
A composition for producing a cell introduction agent, which comprises producing the cell introduction agent by adding the polynucleotide . A composition for producing a cell introduction agent for producing a cell introduction agent, which is used for introducing a polynucleotide into a cell and contains composite particles composed of the polynucleotide and carbonate apatite,
Contains at least phosphate ions and bicarbonate ions,
A composition for producing a cell introduction agent, which comprises producing the cell introduction agent by adding the polynucleotide and calcium ions. The composition for producing a cell introduction agent according to claim 14 or 15, wherein the polynucleotide is DNA and / or RNA . A kit for producing a cell introduction agent for producing a cell introduction agent, which is used for introducing a polynucleotide into a cell and contains composite particles composed of the polynucleotide and carbonate apatite,
A first component containing at least calcium ions;
A second component containing at least phosphate ions and bicarbonate ions,
A cell introduction agent comprising the complex particles is produced by adding the polynucleotide to the first component and then mixing the first component and the second component. For kit.

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