WO2004043495A1

WO2004043495A1 - Delivery agent, method of delivering a target substance to cells, method for producing delivery agent, composition for producing delivery agent, and kit for producing delivery agent

Info

Publication number: WO2004043495A1
Application number: PCT/JP2003/014376
Authority: WO
Inventors: Toshihiro Akaike; Md. Ezharul Hoque Chowdhury; Atsushi Maruyama
Original assignee: The Circle For The Promotion Of Science And Engineering
Priority date: 2002-11-12
Filing date: 2003-11-12
Publication date: 2004-05-27
Also published as: JP4536655B2; AU2003279570A1; JP2006509838A

Abstract

The present invention provides a delivery agent having superior reproducibility and biocompatibility that allows a target substance to be efficiently delivered to cells. In a delivery agent comprising compound particles of DNA and a calcium phosphate-based material, the release time of DNA inside a cell can be controlled to realize efficient cell transfection by adjusting the dissolution rate of the compound particles relative to pH so that at least 50% of the compound particles present at pH 8.0 dissolve within a predetermined time after changing the pH to 6.0.

Description

TITLE OF THE INVENTION

DELIVERY AGENT, METHOD OF DELIVERING A TARGET SUBSTANCE TO CELLS, METHOD FOR PRODUCING DELIVERY AGENT, COMPOSITION FOR PRODUCING DELIVERY AGENT, AND KIT FOR PRODUCING DELIVERY AGENT

TECHNICAL FIELD The present invention relates to a delivery agent for delivering a target substance such as a polynucleotide into a cell, a method of delivering a target substance to cells, a method for producing a delivery agent, a composition for producing a delivery agent, and a kit for producing a delivery agent.

BACKGROUND ART

The delivery of DNA into mammalian cells is an extremely effective research technique relating to gene structure, function and control, and is expected to be useful in fields such as gene therapy and DNA vaccines. Viral methods using a recombinant such as a retrovirus or adenovirus as a vector for gene therapy were typically employed in the prior art for gene delivery.

However, several problems have been pointed out with respect to the use of viruses themselves. Namely, there is the risk of toxicity resulting from the appearance of unexpected mutant viruses, the potential for activity being neutralized in the body due to immunoantigenicity of the virus, limitations on the size of genes that can be used resulting in only genes of a comparatively small size being able to be used, difficulties in production and transport, and high costs.

Consequently, there is considerable activity being conducted for the development of a gene delivery (transfection) technology that does not use viruses to take the place of viral vectors in order to be applicable to basic research and gene therapy. Various non-viral gene delivery methods are known, examples of which include a calcium phosphate method that uses a coprecipitate of DNA and calcium, and a lipofection method that uses compound particles of cationic lipids such as liposomes and anionic DNA.

Development of technology is being actively pursued in order to apply these non-viral gene delivery methods to basic research and gene therapy. However, the efficiency of gene delivery and expression in the case of using non-viral gene delivery methods is still considerably inferior to that in the case of using viral methods.

DISCLOSURE OF THE INVENTION

In consideration of the aforementioned problems, the object of the present invention is to provide a delivery agent for delivering a target substance (such as a drug, protein or polynucleotide) into a cell that has high cell transfection efficiency, superior transfection reproducibility and biocompatibility, a delivery method, a method for producing a delivery agent, a composition for producing a delivery agent, and a kit for producing a delivery agent. As a result of conducting earnest research in order to solve the aforementioned problems, the present inventors found that, in a delivery agent comprising compound particles of DNA and a calcium phosphate-based material, the release time of DNA inside a cell can be controlled to realize efficient cell transfection by adjusting the dissolution rate of the compound particles relative to pH so that at least 50% of the compound particles present at pH 8.0 dissolve within a predetermined time after changing the pH to 6.0, thereby leading to completion of the present invention on the basis of this finding.

Compound particles are taken up into cells by endocytosis, after which they are released into the cytoplasm from endosomes. Since the pH within endosomes is acidic (approximately pH 5), compound particles taken up by endocytosis are subjected to a change in external pH from about pH 7 to pH 5. The present invention realizes extremely high cell transfection efficiency in which compound particles are taken up into cells followed by dissolving rapidly and releasing DNA by adjusting the dissolution rate of the compound particles relative to pH to within a predetermined range.

In the past, studies had been conducted on transfection efficiency in the calcium phosphate method from the viewpoint of how efficiently compound particles are taken up into cells or how efficiently compound particles are able to escape from endosomes after being taken up into cells. However, there have been no examples reported thus far discussing the mechanism when compound particles dissolve and release DNA into the cell after they have been taken up into cells. Namely, the present invention is characterized by controlling the dissolution rate of compound particles relative to pH to allow the compound particles to dissolve rapidly by utilizing the acidic pH present in endosomes after they have been taken up into cells.

Namely, the present invention is as described below.

[1] A delivery agent for delivering a target substance to cells comprising; at least compound particles composed of the target substance and a calcium phosphate-based material; wherein, in the case where pH has been changed from pH 8.0 to pH 6.0, the compound particles have a characteristic that at least 50% of the compound particles present at pH 8.0 dissolve within a predetermined time after changing the pH to 6.0. [2] The delivery agent according to [1], wherein the average particle diameter of the compound particles is 500 nanometers or less.

[3] The delivery agent according to [1] or [2], wherein the calcium phosphate-based material is carbonate apatite.

[4] The delivery agent according to any one of [1] to [3], wherein the target substance is a negatively charged substance.

[5] The delivery agent according to any one of [1] to [4], wherein the target substance is at least one type of substance selected from the group consisting of drug, protein and polynucleotide.

[6] A method of delivering a target substance to cells comprising: a step of delivering the target substance to the cells by using the delivery agent according to any one of [1] to [5].

[7] A method for producing a delivery agent used to deliver a target substance to cells that contains compound particles composed of the target substance and carbonate apatite, comprising: a step of forming the compound particles by preparing a composition at least containing calcium ion, phosphate ion, hydrogen carbonate ion and the target substance.

[8] The method for producing a delivery agent according to [7] comprising: a step of preparing a first solution that contains the calcium ion and the target substance, a step of preparing a second solution that contains the phosphate ion and the hydrogen carbonate ion, and a step of mixing the first solution and the second solution to prepare the composition.

[9] The method for producing a delivery agent according to [7] or [8], wherein the calcium ion concentration of the composition is 0.1 millimolar or more.

[10] The method for producing a delivery agent according to any one of [7] to [9], wherein the phosphate ion concentration of the composition is 0.1 millimolar or more.

[11] The method for producing a delivery agent according to any one of [7] to [10], wherein the hydrogen carbonate ion concentration of the composition is 1.0 millimolar or more. [12] The method for producing a delivery agent according to any one of [7] to [11], wherein the composition additionally contains fluoride ion or strontium ion.

[13] The method for producing a delivery agent according to any one of [7] to [12], wherein the pH of the composition is pH 6.0 to pH 9.0. [14] The method for producing a delivery agent according to any one of [7] to [13], wherein the compound particles are formed by holding the composition at 10°C or higher.

[15] A composition for producing a delivery agent used to deliver a target substance to cells that contains compound particles composed of the target substance and carbonate apatite; wherein, the composition contains at least calcium ion, phosphate ion and hydrogen carbonate ion, and the delivery agent is produced by adding the target substance to the composition. [16] A composition for producing a delivery agent used to deliver a target substance to cells that contains compound particles composed of the target substance and carbonate apatite; wherein, the composition contains at least phosphate ion and hydrogen carbonate ion, and the delivery agent is produced by adding the target substance and calcium ion to the composition.

[17] A kit for producing a delivery agent used to deliver a target substance to cells that contains compound particles composed of the target substance and carbonate apatite; wherein, the kit is composed of: a first component containing at least calcium ion, and a second component containing at least phosphate ion and hydrogen carbonate ion, and the delivery agent containing the compound particles is produced by adding the target substance to the first component followed by mixing the first component and the second component.

A target substance can be delivered extremely efficiently into a cell by using the delivery agent of the present invention. Since the compound particles contained in the delivery agent of the present invention have the characteristic of at least 50% of the compound particles dissolving when the pH is changed from pH 8.0 to pH 6.0, after having been taken up into a cell, they rapidly release the target substance. In addition, various substances can be delivered into cells, ranging from low molecular weight drugs to polyanions having a comparatively large molecular weight such as DNA. In addition, since the compound particles contained in the delivery agent of the present invention are composed of a calcium phosphate-based material, they are easily taken up into cells and there is little concern over toxicity.

In addition, the efficiency by which the aforementioned compound particles are taken up into cells can be further enhanced by making the average particle diameter of the compound particles 500 nanometers or less. Moreover, cell uptake efficiency can also be further enhanced by using carbonate apatite as the aforementioned calcium phosphate-based material to inhibit growth into large crystals. In addition, the use of drug, protein or polynucleotide as the aforementioned target substance enables the present invention to be suitably used for the treatment of disease, gene recombination techniques, RNA interference and so forth.

In addition, the delivery method of the present invention allows a target substance to be delivered extremely efficiently into a cell since it uses the delivery agent of the present invention. Thus, it can be suitably used for the treatment of disease, gene recombination techniques, RNA interference and so forth.

Further, the delivery agent production method of the present invention is a method for producing a delivery agent that contains compound particles composed of a target substance and carbonate apatite by preparing a composition consisting of at least calcium ion, phosphate ion, hydrogen carbonate ion and a target substance, and is able to easily produce the delivery agent of the present invention. In addition, in the delivery agent production method of the present invention, the formation efficiency of the compound particles can be enhanced by preparing the aforementioned composition by preparing a first solution containing calcium ion and a target substance, preparing a second solution separate from the first solution that contains phosphate ion and hydrogen carbonate ion, and mixing the first solution and the second solution. In addition, the formation efficiency of the aforementioned compound particles can be enhanced by making the calcium ion concentration of the aforementioned composition 0.1 millimolar or more. In addition, the formation efficiency of the aforementioned compound particles can be enhanced by making the concentration of phosphate ions of the aforementioned composition 0.1 millimolar or more. In addition, the formation efficiency of the aforementioned compound particles can be enhanced by making the concentration of hydrogen carbonate ions of the aforementioned composition 1.0 millimolar or more. In addition, a delivery agent having a high cell transfection efficiency can be produced by controlling the pH of the aforementioned composition to within the range of pH 6.0 to pH 9.0. In addition, a delivery agent having a high cell transfection efficiency can be produced by maintaining the temperature of the aforementioned composition at 10°C or higher.

Further, since the composition for producing a delivery agent of the present invention contains at least calcium ion, phosphate ion and hydrogen carbonate ion, the delivery agent of the present invention can be easily prepared simply by adding a target substance at the time of use. In addition, since another composition for producing a delivery agent of the present invention contains at least phosphate ion and hydrogen carbonate ion, the delivery agent of the present invention can be easily prepared simply by adding a target substance and calcium ion at the time of use.

Moreover, since the delivery agent production kit of the present invention is composed of a first component containing at least calcium ion and a second component containing at least phosphate ion and hydrogen carbonate ion, a delivery agent capable of realizing high transfection efficiency can be easily prepared simply by adding a target substance to the aforementioned first component and mixing the first component and the second component.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 (A) is an infrared spectra of generated carbonate apatite.

Fig. 1(B) is an infrared spectra of generated hydroxyapatite. Fig. 1(C) is an infrared spectra of generated hydroxyapatite, incubated with HCO₃- -buffered DMEM at 37°C for 4 hrs

Fig. 1(D) is a X-ray diffraction patterns of carbonate apatite. Fig. 1 (E) is a X-ray diffraction pattern of hydroxyapatite. Fig. 1(F) is a X-ray diffraction pattern of fluoridated carbonate apatite containing 0.65% fluoride ion.

Fig. 1(G) is X-ray diffraction pattern of fluoridated carbonate apatites, containing 1.43% fluoride ion. Fig. 1(H) is X-ray diffraction pattern of fluoridated carbonate apatites, containing 2.5% fluoride ion.

Fig. 2(A) is a graph of transfection efficiency when gene delivery was carried out using HeLa cells in a medium containing 10% FBS while changing the pH during formation of DNA/carbonate apatite particles. Fig. 2(B) is a graph of transfection efficiency when gene delivery was carried out using HeLa cells in a medium containing 10% FBS while changing the temperature during formation of DNA/carbonate apatite particles.

Fig. 2(C) is a graph of the levels of gene expression when gene delivery was carried out by carbonate apatite, hydroxyapatite and lipofectamine using HeLa cells.

Fig. 2(D) is a graph of the levels of gene expression when gene delivery was carried out by carbonate apatite, hydroxyapatite and lipofectamine using HepG2 cells.

Fig. 2(E) is a graph of the levels of gene expression when gene delivery was carried out by carbonate apatite, hydroxyapatite and lipofectamine using NIH3T3 cells.

Fig. 2(F) is a graph of the levels of gene expression when gene delivery was carried out by carbonate apatite, hydroxyapatite and lipofectamine using mouse primary hepatocytes cells. Fig. 2(G) illustrates the results of examining a survival rate of HeLa cells using MTT assay.

Fig. 3(A) is a photograph of a scanning electron micrograph (scale bar: 600 nanometers) of carbonate apatite-mediated cellular uptake of DNA.

Fig. 3(B) are photographs of the cellular uptake of Pl-labeled plasmid DNA associated with carbonate apatite and hydroxyapatite. (a) no uptake of DNA (control), since endocytosis was blocked by energy depletion (50 mM 2-deoxy glucose and 1 mM Na-azide). DNA/carbonate apatite particles were prepared in 1 ml serum-free media using 6mM Ca²⁺ and 2 ]g DNA. 40 ng (b) and 200 ng (c) of DNA found, respectively, in 20 II and 100 II of 1ml suspension, were allowed for cellular uptake for 4 hrs. (d), 2 ig of DNA adsorped to hydroxyapatite was allowed for uptake for the same period of time.

Fig. 3(C) are photographs of the endosomal escape of endocytosed Pl-labeled DNA, as evident after colocalization with a fluorescence probe (Lyso-Sensor) for endosomes.

Fig. 4(A) is a graph of effect of bafilomycin A1 (an inhibitor of v-ATPase) on transfection. Cells were incubated with DNA/carbonate apatite particles and 200 nM bafilomycin A1 for 6 hours. After washing with 5 mM EDTA in PBS, cells were grown for 1 day and luciferase expression was detected.

Fig. 4(B) is a graph of the changes in luciferase expression for increasing concentrations of F- (0.01 to 3 mM) and strontium (0.01 to 3 mM) added during generation of DNA/carbonate apatite particles.

Fig. 4(C) are photographs of the uptake of Pl-labeled plasmid DNA (pEGFP-N2) and GFP expression for carbonate apatite (top row) and fluoridated carbonate apatite (bottom row), as observed after 20 hours following 4 hours incubation with the particles and treatment with 5 mM EDTA in PBS.

Fig. 4(D) is a graph of the changes in luciferase expression for 1M concentrations of F- added during formation of DNA/carbonate apatite particles. After incubation of cells with the particles, cells were washed with EDTA and grown for 1 day, as described above.

Fig. 5(A) is a graph of the pH-dependent dissolution behaviors of apatites. Dissolution rates (at pH 7.0) of fluoridated carbonate apatites prepared by addition of 0-3 mM F- during generation of carbonate apatite at pH 7.5 (described in experimental protocol), were studied by turbidity measurement at 320 nm of apatite suspensions just after being adjusted to the pH 7.0 with 1 N HCl.

Fig. 5(B), is a graph of the dissolution rates of carbonate apatite, fluoridated carbonate apatite and strontium-containing carbonate apatite with decreasing pH from 7.5 to pH 7.0 or pH 6.8.

Fig. 5(C) is a graph of the degree of solubilization of carbonate apatite, fluoridated carbonate apatite and hydroxyapatite powders in acetate buffers of pHs 6.5 and 5.5 , as estimated by % of Ca²⁺ released from the apatites. Fig. 5(D) are photographs of the intracellular release of Pl-labeled plasmid DNA from apatite crystals, assessed by observed fluorescence intensity of PI. Cells were incubated with DNA/carbonate apatite particles (a, d) for 4 hr and after EDTA treatment, incubated for more 5 hr (b, c, e) either in absence (b, e) or presence of 200 nM bafilomycin A (c). Similarly cells were incubated with DNA/fluoridated carbonate apatite particles for 4 hr (f), treated with EDTA and incubated for more 5 hr (g). White and pink bars indicate 20 l m and 50 1m, respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

Exemplary embodiments of the present invention are described below with the following order.

[I] Delivery agent

[II] Delivery agent Production Method Delivery Method

[I] Delivery agent

The delivery agent of the present invention is used to deliver a target substance to a cell, and contains compound particles composed of at least a target substance and a calcium phosphate-based material. The compound particles contained in the delivery agent of the present invention have the characteristic of at least 50% of the compound particles present at pH 8.0 dissolving within a predetermined amount of time after the pH is changed to pH 6.0 in the case the pH has been changed from 8.0 to 6.0. In the specification of the present application, the property by which the compound particles dissolve as a result of changing the pH from that higher than pH 7 to pH 7 or lower will be referred to as "pH solubility".

Since the pH within endosomes is acidic (about pH 5), although compound particles taken up by endocytosis are exposed to a change in external pH from about pH 7 to pH 5, the compound particles of the present invention release a target substance as a result of rapidly dissolving after being taken up into cells due to their high pH solubility. As a result, the target substance can be efficiently delivered to cells.

In the present invention, although it is sufficient that the pH solubility required of the compound particles be of a level such that 50% or more of the compound particles present at pH 8.0 dissolve within a predetermined amount of time as a result of changing the pH from pH 8.0 to pH 6.0, the higher the pH solubility of the compound particles in the present invention, the better it is. The degree of pH solubility of the compound particles is defined by the three parameters of (1) the amount of the change in pH required for the compound particles to dissolve, (2) the amount of time required for the compound particles to dissolve, and (3) the percentage of compound particles that dissolve due to a change in pH. Namely, this means that the smaller the change in pH required for the compound particles to dissolve, the higher the pH solubility of the compound particles. In addition, this also means that the shorter the time required for the compound particles to dissolve, the higher the pH solubility of the compound particles. In addition, this also means that the higher the percentage of compound particles that dissolve due to a change in pH, the higher the pH solubility of the compound particles.

In the present invention, the change in pH required for 50% or more of the compound particles to dissolve is such that the starting pH value is from greater than pH 7.0 up to and including pH 8.0, and the pH value after the change in pH is from 6.0 up to and including 7.0. In the present invention, the smaller the size of this pH change the better. In the present invention, the pH value before the change in pH is from greater than pH 7.0 up to and including pH 8.0, preferably greater than pH 7.0 up to and including pH 7.5, more preferably greater than pH 7.0 up to and including pH 7.2, still more preferably greater than pH 7.0 up to and including pH 7.1 , particularly preferably greater than pH 7.0 up to and including pH 7.05, and most preferably greater than pH 7.0 up to and including pH 7.01. In addition, the pH value after the change in pH is from pH 6.0 up to and including pH 7.0, preferably from pH 6.5 up to and including pH 7.0, more preferably from pH 6.7 up to and including pH 7.0, still more preferably from pH 6.8 up to and including pH 7.0, even more preferably from pH 6.9 up to and including pH 7.0, and particularly preferably from pH 6.95 up to and including pH 7.0. In addition, the time required for 50% or more of the compound particles to dissolve is preferably within 10 minutes, more preferably within 5 minutes, still more preferably within 2 minutes, and particularly preferably within 1 minute.

In addition, the percentage of compound particles that dissolve due to a change in pH is 50% or more, preferably 80% or more and more preferably 100%, of the compound particles present before the change in pH.

Namely, the compound particles used in the present invention preferably have the characteristic of rapidly dissolving due to a slight change in pH towards the acidic side. Such compound particles are composed of a target substance and a calcium phosphate-based material. Any target substance can be used without any particular restrictions provided it is a substance that is capable of forming compound particles with the calcium phosphate-based material. More specifically, polyanions of drugs, proteins, polynucleotides and so forth can be used. In addition, negatively charged substances are preferably used as target substances in the present invention.

Specific examples of drugs that can be used in the present invention include antitumor drugs and antitumor antibiotics. Examples of antitumor drugs include Methoterxate (antifolate), Vinblastine (vinca alkaloid), and Antracyclines (Daunomycin, Adriamysin). Examples of antitumor antibiotics include Duocarmycin, Enediynes, Neocarzinostatin, Calicheamicin, and Macrolide. Since the cellular uptake efficiency of the drug can be improved by forming compound particles using such drugs, they can be suitably used for the treatment of various diseases. Examples of polynucleotides that can be used include DNA, RNA as well as mixed polynucleotides composed of DNA and RNA. For example, in the case of carrying out gene recombination using the delivery agent of the present invention, compound particles should be formed using vector DNA that contains the gene desired to be expressed. Here, any form of DNA may be used, including cyclic plasmid DNA, linear plasmid DNA, artificial chromosomes and triplex DNA. Alternatively, compound particles may also be formed using RNA capable of regulating cell function, examples of which include antisense RNA and siRNA that causes RNA interference.

In the present invention, the calcium phosphate-based material that composes the compound particles is a material having Ca and PO₄ for its main components. In the present invention, the calcium phosphate-based material is preferably a kind of apatite. Although examples of apatites that can be used include hydroxyapatite and carbonate apatite, carbonate apatite is used particularly preferably. The carbonate apatite preferably used in the present invention is represented by the composition formula Caio-m m(P0₄)6(CO₃)₁-nYn- Here, X is an element that is able to partially replace the Ca in the aforementioned composition formula, examples of which include Sr, Mn and rare earth elements, m is a positive number from 0 to 1 , preferably from 0 to 0.1 , more preferably from 0 to 0.01 , and particularly preferably from 0 to 0.001. In addition, Y is a unit capable of partially replacing CO₃ in the aforementioned composition formula, examples of which include OH, F and CI. n is a positive number from 0 to 0.1 , preferably from 0 to 0.01 , more preferably from 0 to 0.001 , and particularly preferably from 0 to 0.0001. The average particle diameter of the compound particles contained in the delivery agent of the present invention is preferably 500 nanometers or less, more preferably 400 nanometers or less, still more preferably 300 nanometers or less, and particularly preferably 200 nanometers or less. The smaller the average particle diameter of the compound particles, the greater the improvement in uptake efficiency of the compound particles into cells. Although there are no particular restrictions on the lower limit of the average particle diameter of the compound particles, it is normally 20 nanometers or more.

The delivery agent of the present invention contains the aforementioned compound particles. There are no particular restrictions on the drug form of the delivery agent of the present invention provided it can be delivered to cells without causing a change in the target substance, and may be in any form such as a powder, solid or liquid. [II] Delivery agent Production Method Next, an explanation is provided of a method for producing the delivery agent of the present invention. The delivery agent production method of the present invention is a method that is characterized by forming the aforementioned compound particles by preparing a composition that at least contains as essential components the four components consisting of calcium ion, phosphate ion, hydrogen carbonate ion and a target substance. This composition is preferably prepared in the form of an aqueous solution. Here, the calcium ion concentration in the composition is preferably 0.1 millimolar or more, more preferably 0.5 millimolar or more, and still more preferably 1 millimolar or more. In addition, the calcium ion concentration is preferably 1 molar or less, more preferably 100 millimolar or less, and still more preferably 10 millimolar or less.

The phosphate ion concentration in the composition is preferably 0.1 millimolar or more, more preferably 0.5 millimolar or more, and still more preferably 1 millimolar or more. In addition, the phosphate ion concentration is preferably 1 molar or less, more preferably 100 millimolar or less, and still more preferably 10 millimolar or less.

The hydrogen carbonate ion concentration in the composition is preferably 1.0 millimolar or more, more preferably 5 millimolar or more, and still more preferably 10 millimolar or more. In addition, the hydrogen carbonate ion concentration is preferably 10 molar or less, more preferably 1 molar or less, and still more preferably 100 millimolar or less.

Although there are no particular restrictions on the manner in which these ions are supplied to the composition, salts of a calcium ion source, phosphate ion source and hydrogen carbonate ion source are preferably added in an aqueous solution. The amounts added are preferably controlled so that the calcium ion concentration, phosphate ion concentration and hydrogen carbonate ion concentration are within the aforementioned ranges.

In addition, although the concentration of target substance in the composition is normally preferably 1 μg/L to 1 mg/L, the concentration may exceed or fall below this range as long as the target substance can be delivered into the cells.

There are no particular restrictions on the order in which the aforementioned four components are mixed, and although the composition may be prepared by mixing in any order, one example of a preferable preparation method consists of preparing a first solution containing calcium ion and a target substance, preparing a separate second solution containing phosphate ion and hydrogen carbonate ion, and then mixing the first solution and the second solution to prepare a composition containing the four essential components. The total amount of calcium ion to ultimately be contained in the composition is not required to be contained in the first solution. Namely, a portion of calcium ion to be ultimately contained in the composition may be contained in the first solution, while the remainder of the calcium ion may be contained in the second solution.

In the case of setting the calcium ion concentration of the composition comparatively high, there are cases in which a precipitate of carbonate apatite forms prior to formation of a complex with the target substance when the target substance is finally added. However, compound particles can be formed efficiently by mixing a first solution containing calcium ion and the target substance with a second solution containing phosphate ion and hydrogen carbonate ion. In addition, the Ca or CO₃ in the carbonate apatite may be partially substituted in the aforementioned composition by adding fluoride ion, chorine ion, Sr or Mn and so forth. The amount of fluoride ion, chlorine ion, Sr or Mn added is to be within a range that does not significantly effect the pH solubility or particle diameter range of the compound particles formed.

In addition, other components can also be added to the composition within a range that does not deviate from the object of the present invention of forming compound particles.

For example, the aforementioned composition may be prepared by using a medium for culturing the cells targeted for delivery of the target substance. Normally, the experimental procedure becomes complex since it is necessary to adjust the medium to ultimately obtain the intended medium composition by calculating the concentration of each component contained in the delivery agent in advance in the case the composition of the medium changes and the change in the composition is large when the delivery agent is added to the medium. However, by preparing a composition that contains the aforementioned four essential components in advance by using a liquid medium, forming the compound particles in this liquid medium and then using this as a delivery agent, large changes in the composite ratio of the components of the medium caused by addition of delivery agent can be prevented.

In the case of preparing the aforementioned composition using a medium, the medium is first prepared in the form of a liquid medium. A composition containing the four essential components is then prepared by supplementing the medium with those essential components, consisting of calcium ion, phosphate ion, hydrogen carbonate ion and target substance, which are not contained in the liquid medium or are not contained at the required concentrations.

In addition, in order to prepare the aforementioned composition easily, a composition for producing a delivery agent can be provided that allows the aforementioned composition to be prepared simply by adding a substance that is to be delivered to the cells. This type of composition for producing a delivery agent is provided in the form of a composition that contains at least the three components of calcium ion, phosphate ion and hydrogen carbonate ion at a predetermined ratio. At the time of use, a composition can be easily prepared that contains the four essential components of calcium ion, phosphate ion, hydrogen carbonate ion and target substance if a predetermined amount of target substance to be delivered to the cells is added to this composition for producing a delivery agent. In addition, a composition for producing a delivery agent can also be provided that allows the aforementioned composition to be prepared simply by adding a target substance to be delivered to the cells and calcium ion. This type of composition for producing a delivery agent is provided in the form of a composition containing at least the two components of phosphate ion and hydrogen carbonate ion at a predetermined ratio. At the time of use, a composition can be easily prepared that contains the four essential components of calcium ion, phosphate ion, hydrogen carbonate ion and target substance if predetermined amounts of target substance and calcium ion are added to this composition for producing a delivery agent. This type of composition may be provided in the form of a solution, for example, or provided in the form of a powder or paste. In addition, each of the ions may be contained in the form of salts in this composition for producing a delivery agent.

Moreover, another mode for easily preparing the aforementioned composition may be provided in the form of a delivery agent production kit composed of a first component that contains at least calcium ion, and a second component that contains at least phosphate ion and hydrogen carbonate ion at a predetermined ratio. At the time of use of this delivery agent production kit, by mixing the aforementioned target substance with the first component followed by mixing the first component with the second component, a composition can be easily prepared that contains the four essential components of calcium ion, phosphate ion, hydrogen carbonate ion and target substance. Here, the first and second components may be respectively provided in the form of solutions, or they may be provided in the form of powders or pastes. In addition, each of the ions may be contained in the form of salts in the first and second components.

After a composition containing the four essential components of calcium ion, phosphate ion, hydrogen carbonate ion and target substance has been prepared, if this composition is allowed to stand for predetermined amount of time, compound particles are formed that are composed of the target substance and carbonate apatite.

Here, the compound particles are preferably formed while maintaining the temperature of the composition at 10°C or higher, more preferably 25°C or higher, still more preferably at 37°C or higher, and particularly preferably at 50°C or higher. In addition, although there are no particular restrictions on the upper limit of the temperature provided it is within a range that does cause degeneration of the target substance, it is normally 80°C or lower, and may preferably be 70°C or lower.

In addition, the compound particles are preferably formed by adjusting the pH of the composition to pH 6.0 to 9.0, more preferably to pH 7.0 or higher, still more preferably to pH 7.1 or higher, particularly preferably to pH 7.2 or higher, and most preferably to pH 7.5 or higher. In addition, the compound particles are preferably formed by also adjusting the pH of the composition to pH 9.0 or below, more preferably to pH 8.5 or below, and particularly preferably to pH 8.0 or below.

After preparing the aforementioned composition, the composition is allowed to stand until the compound particles are formed. The amount of time required for the compound particles to form is normally from about 1 minute to 24 hours, while in many cases, compound particles are formed that can be observed microscopically in about 10 minutes to 1 hour.

A composition containing these compound particles can be used directly as a delivery agent. In addition, it may also be used as a powder by isolating the compound particles from the composition, or it may be used as a delivery agent after transforming the compound particles into a solid form such as tablets.

[Ill] Delivery Method

The delivery method of the present invention is characterized by delivering a target substance into cells using the delivery agent of the present invention. Here, the target substance delivered to the cells is preferably a negatively charged substance capable of regulating cell function. In the present invention, various types of cells including bacterial cells, actinomyces cells, yeast cells, mold cells, plant cells, insect cells and mammalian cells can be used as cells targeted for delivery of the target substance. Among these, animal cells, and particularly mammalian cells, can be used preferably. The cells targeted for delivery of a target substance include both in vitro and in vivo cells. Namely, any cells such as cultured cells, cultured tissue or biological specimens may be used.

In the case of using cultured cells, a target substance can be delivered into the cells by preparing a medium that contains the delivery agent of the present invention, and culturing using this medium under ordinary culturing conditions.

In addition, in the case of using the delivery agent of the present invention as a pharmaceutical for treatment of various diseases, a substance having pharmacological activity in cells of the body can be delivered directly by, for example, preparing a delivery agent containing compound particles composed of a substance having pharmacological activity and a calcium phosphate-based material, and administering it to mammals (including humans) subcutaneously, intramuscularly, intraperitoneally or intravenously and so forth. In addition, in the case of using as a pharmaceutical for gene therapy, a delivery agent can be prepared that contains compound particles composed of polynucleotide capable of regulating cell function (e.g., vector DNA, antisense RNA or RNAi) and a calcium phosphate-based material, and then delivering to and expressing in the target cells. Examples of diseases eligible for gene therapy include cancer and genetic diseases. Examples

Hereinafter, the present invention is further explained in more detail with reference to the following examples. It is to be understood that the present invention is not limited to the examples.

The reagents used in the examples are indicated below. pGL3 (Promega), which contains luciferase under the control of SV40 promoter, and pEGFP-N2 (Clontech), which contains GFP gene under the control of CMV, were propagated with XL-1 Blue (by a method indicated in Molecular Cloning) and purified with the Qiagen Plasmid Kit. Propidium iodide (PI) and

LysoSensor™ Green DND-189 were purchased from Sigma and Molecular Probes, respectively. Lipofectamine and DMEM (catalog no. 12800) were purchased from Gibco BRL.

In addition, cell lines HeLa, HepG2 and NIH3T3 were cultured under the atmosphere of 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL) containing 10% fetal bovine serum (FBS), 50 μg/ml of penicillin, 50 μg/ml of streptomycin and 100 μg/ml of neomycin in a 75 square centimeters flask. Primary hepatocytes were isolated from the livers of male ICR mice (5-7 weeks old) (SLC, Shizuoka, Japan) by using a modified version of the in situ perfusion method previously described, seeded in collagen coated 24-well plate , and cultured in the same manner using Williams' E (WE) medium (Gibco BRL) instead of DMEM.

Example 1 Delivery agent [1] Preparation of Delivery agent 3 to 6 II of 1 M CaCI₂ was mixed with 2 Ig of plasmid DNA in 1 ml of fresh serum-free HCO₃ ^" - buffered (pH 7.5) medium (DMEM or WE) and incubated for 30 min at 37°C for complete generation of DNA/carbonate apatite compound particles.

The compositions of the DMEM medium and WE medium used are indicated to follow. Table 1 DMEM medium

Table 2 WE medium

For generation of fluoridated or strontium- containing carbonate apatite, 0.01 to 3 II of 1 M NaF or SnCI₂ was added along with Ca²⁺ and DNA and incubated as described above.

Addition of only 3 mM Ca²⁺ to the HCO₃ ^" buffered cell culture medium (DMEM or Willium E, pH 7.5) and incubation at 37°C for 30 min, resulted in microscopically visible particles. Generation of these particles only in HCO₃ ^", but not in Hepesbuffered media or solution (pH 7.5) containing the same amount of total Ca²⁺ (4.8 mM) and phosphate (0.9 mM), indicates the possible involvement of carbonate along with phosphate and Ca²⁺ in particle formation.

[2] Analysis of Formed Particles

The formed particles were analyzed by chemical analysis, infrared spectroscopy and X-ray diffraction. (Chemical Analysis) Following generation of carbonate apatite as described above, using 6 mM Ca²⁺ and no DNA, precipitated particles were lyphilized after centrifugation and washing with distilled deionized water. Other apatite particles generated as described above, were also similarly lyphilized. Calcium and phosphorus contents were determined using SPS 1500 VR Atomic Absorption Spectrophotometer. Carbon and fluorine were estimated by CHNS-932 (Leco, USA) and SX-elements micro analyser, YS-10 (Yanaco, Japan), respectively. (Infrared Spectroscopy)

Fourier transform-infrared spectroscopy of apatite particles prepared as described above, was performed using FT/IR-230, JASCO. The samples were ground in a mortar and approximately 1 mg was thoroughly mixed with 300 mg of ground spectroscopic grade KBr. Transparent pellets were prepared in a KBr die with an applied load of 8000 kg, under a vacuum of 0.5 torr. (X-Ray Diffraction) X-ray powder analysis of the prepared particles was performed using the M18XHF-SRA diffraction system.

Elemental analysis proved the existence of C (3%), P (17%) and Ca²⁺ (32%) and FT-IR spectra (Fig. 1(A)) identified carbonate, as evident from the peaks between 1410 and 1540 cm^"1 and at approximately 880 cm^"1, along with phosphate in the particles, as shown by the peaks at 1000-1100 cm^"1 and 550-650 cm^"1 (Fig. 1(B)). X-ray diffraction patterns (Fig. 1 (C)) indicated less crystalline nature, represented by broad diffraction peaks of the particles, compared to that of hydroxyaptite (Fig. 1(D)) - an intrinsic property of carbonate apatite.

Example 2 Gene Delivery to Cells

[1] Measurement of Luciferase Expression Level

The expression level of luciferase was measured using the delivery agent of the present invention in order to investigate the transfection efficiency of the delivery agent of the present invention prepared according to part [1] of Example 1.

Cells from the exponentially growth phase were seeded at 50,000 cells per well into 24-well plates the day before transfection. Medium with generated DNA-containing particles was added with 10% FBS to the rinsed cells.

After 4 hr incubation, the medium was replaced with serum supplemented medium and the cells were cultured for 1 day. Luciferase gene expression was monitored by using a commercial kit (Promega) and photon counting (TD-20/20 Luminometer, USA). Each transfection experiment was done in triplicate and transfection efficiency was expressed as mean light units per mg of cell protein.

Primary hepatocytes were transfected 3 hr after seeding into collagen coated 24-well plates. Transfection by calcium phosphate-DNA co-precipitation was performed according to Jordan M, et al (Jordan, M., Schallhom, A. & Wurm FM. Transfecting mammalian cells:optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic acids research 24, 596-601 (1996)). Briefly, 12 ig of plasmid DNA was added to 300 II of a solution containing 250 mM CaCI₂. This solution was added to 30011 of a 2^χHBS (50 mM Hepes, 140 mM NaCI, 1.5 mM Na₂HPO₄.2H₂O, pH 7.05) and mixed rapidly by gentle pipetting twice.

The DNA/CaPi mixture was incubated at room temperature for the period of time indicated. After addition of 100 II of the incubated mixture dropwise to 1 ml serum supplemented media of each well, cells were incubated for 4 hr and like above, after replacement with fresh serum media, grown for 1 day.

In order to investigate the effects of calcium ion concentration, pH of the hydrogen carbonate ion-buffered medium and incubation temperature, transfection efficiency was evaluated for gene delivery using HeLa cells in medium containing 10% FBS while changing the calcium ion concentration and incubation temperature.

Fig. 2(A) illustrates the transfection efficiency when the calcium ion concentration added to DMEM was changed during formation of DNA/carbonate apatite particles dependent on the pH of hydrogen carbonate ion-buffered medium, while Fig. 2(B) illustrates the transfection efficiency when the temperature was changed during formation of DNA/carbonate apatite particles dependent on the pH of hydrogen carbonate ion-buffered medium. Here, transfection efficiency was examined based on the expression level of luciferase during gene delivery using HeLa cells in medium containing 10% FBS.

Generation of carbonate apatite particles was governed by Ca²⁺concentrations, pH of the HCO₃ ^" buffered media and incubation temperatures. The optimal Ca²⁺ concentration required for generation of effective amount of DNA/carbonate apatite particles for obtaining high transfection efficiency, was inveresely related to the pH of the media (Fig. 2(A)) and the incubation temperature (Fig. 2(B)). The decline below the high efficiency level of transfection was due to the formation of low amount of the particles (microscopically observed), since increase in pH, temperature and / or the Ca²⁺ content of aqueous medium, contributed to the development of solution supersaturation- a prior need for generation of the particles. By simply controlling all of the parameters for particle formation, for the first time the present inventors could develop a flexible transfection system with highly reproducible outcome.

To evaluate the role of carbonate apatite as a powerful carrier of genetic material, the present inventors compared transfection efficiency of different techniques including two frequently used ones- CaP co-precipitation method (mentioned above) and lipofection.

Fig. 2(C) illustrates the gene expression levels when gene delivery was performed with carbonate apatite, hydroxyapatite and lipofectamine using HeLa cells, Fig. 2(D) illustrates the gene expression levels when gene delivery was performed with carbonate apatite, hydroxyapatite and lipofectamine using HepG2 cells, Fig. 2(E) illustrates the gene expression levels when gene delivery was performed with carbonate apatite, hydroxyapatite and lipofectamine using NIH3T3 cells, and Fig. 2-6 illustrates the gene expression levels when gene delivery was performed with carbonate apatite, hydroxyapatite and lipofectamine using mouse primary hepatocytes.

Here, DNA carbonate apatite particles were generated by addition of 3 to 6 mM Ca²⁺ (3 mM Ca²⁺ in all the cases where Ca²⁺ concentration was not specified) and 2 ig plasmid DNA to 1 ml HCO₃ ^" -buffered medium (pH 7.5), followed by incubation for 30 minutes at 37°C. In the case of the HeLa cells illustrated in Fig.2(C), 6 mM Ca²⁺ was added along with 2 ig DNA to generate particles in 1 ml serum-free media (described above) and 100 II (200 ng DNA) and 20 II (40 ng DNA) of 1 ml suspension were applied for transfection in presence of FBS. DNA/hydroxyapatite particles were generated according to Jordan, M., at al. DNA/lipofectamine complex was prepared at 1 :6 weight ratio according to the manufacturer's protocol. Transfection of cells was performed in the same manner as mentioned in experimental protocol and 2 1 g DNA was used (if not specified elsewhere) in each of the wells of tissue culture plate during transfection by different methods. In HeLa cell, for example, luciferase expression level for carbonate apatite-mediated transfection was over 15- and 25-fold higher than for lipofection and CaP co-precipitation method, respectively (Fig. 2(C)). Nano gram level of DNA was even sufficient for efficient transgene expression (Fig. 2(C)). Transfection efficiency was also significantly high in HepG2 (Fig. 2(D)), NIH 3T3 cells (Fig. 2(E)) and mouse primary hepatocytes (Fig. 2(F)).

Treatment of the cells with EDTA after 4 hr of DNA uptake resulted in over 2 fold reduction in luciferase expression level (Fig. 2(C)), which suggests that DNA uptake for a longer period, contributed to the efficient gene expression and compensated for DNA hydrolysis by nuclease, as also clarified from the persistence of fluorescence intensity of Pl-labeled DNA (not illustrated). [2] MTT Assay

An MTT assay was conducted using HeLa cells to verify that the appearance of high transfection efficiency was not the result of a high cell survival rate.

HeLa cells were transfected and cultured for 1 day as described above. 30 II of MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution (5mg/ml) was added to each well and incubated for 4 hrs. 0.5 ml of DMSO was added after removal of media. After dissolving crystals and incubating for 5 min at 37°C, absorbance was measured in a microplate reader at 570 nm with a reference wavelength of 630 nm.

Fig. 2(G) illustrates the results of examining the survival rate of HeLa cells using the MTT assay. [3] Analysis of Compound Particles Contained in Delivery agent (Scanning Electron Microscope (SEM))

Liquid droplets of a mixture of DNA and carbonate apatite prepared in the manner described in the gene delivery protocol were added to the carbon-coated stage of an SEM, allowed to dry and then observed with the SEM (S-800, Hitachi, Japan). Fig. 3(A) illustrates the resulting scanning electron microscope micrograph. The scale bar is 600 nanometers.

Carbonate, when present in the apatite structure, limits the size of the growing apatite crystals and increases the dissolution rate. The present inventors carried out scanning electron microscopic observation of generated carbonate apatite (Fig. 3(A)) which revealed reduced growth of the crystals, most of which had diameters of 50 to 300 nm. (Confocal Laser Microscope)

The present inventors verified this size limiting effect of carbonate by observing cellular uptake of the PI (propidium iodide)-labeled plasmid DNA adsorbed to the apatites, since large particles are phagocytosed less efficiently than small ones. pGL3 vector was labeled with PI at a PI/DNA ratio of 1 :1 and particles generated with this labeled plasmid (described in transfection protocol), were incubated with HeLa cells for 6 hours. Acidic compartments were labeled with 51 M LysoSensor, according to the instructions provided by Molecular Probes, and membrane-bound precipitates were removed by 5 mM EDTA in PBS before observation by LEICA TCS-NT.

Fig. 3(B) depicts Apatite-mediated cellular uptake, release and expression of DNA. In Fig. 3(B), uptake of DNA is not observed in (a)

(control experiment). This is because endocytosis was blocked by energy depletion (50 mM 2-deoxy glucose and 1 mM Na-azide). DNA/carbonate apatite particles were prepared in 1 ml serum-free media (described in legend to Fig. 2) using 6mM Ca²⁺ and 2 ig DNA. 40 ng (b) and 200 ng (c) of DNA in 20 II and 100 11 of 1ml suspension respectively, were allowed for cellular uptake for 4 hr. In (d), 2 micrograms of DNA were adsorbed onto hydroxyapatite and taken up into the cells for the same amount of time.

DNA was carried into the cells by carbonate apatite (Fig. 3(B)(c)) at least 10 times more efficiently than hydroxyapatite, generated by 1 min incubation (Fig. 3(B)(d)).

Longer period (30 min) incubation resulted in large hydroxyapatite particles, showing significantly reduced transfection efficiency (Fig. 2(C)) due to extremely low cellular uptake of DNA (not shown here).

Our findings, therefore, clearly suggest that carbonate apatite is superior over hydroxyapatite for its intrinsic property of preventing crystal growth, leading to high efficiency cellular uptake of DNA.

To evaluate the role of endosomal escape of DNA in transgene expression, following endocytosis of Pl-labeled plasmid DNA, the present inventors labeled endosomes with LysoSensor (a fluorescence probe for endosomes). Following 6 hr of DNA uptake by cells, a significant portion of DNA appeared to be released from endosomes after colocalization of plasmid DNA with endosomes (see Supplementary Fig.3(C)). [4] Effect of pH on Transfection

In order to investigate the effect of pH on transfection, changes in transfection efficiency were investigated using bafilomycin A1 , a specific inhibitor of v-ATPase (a proton pump used to acidify endosomes). HeLa cells were incubated with DNA/carbonate apatite particles and 200 nM bafilomycin A1 for 6 hours. After washing with 5 mM EDTA in PBS, cells were grown for 1 day and luciferase expression was detected. Those results are shown in Fig. 4(A). Treatment with bafilomycin A1 , a specific inhibitor of v-ATPase (a proton pump for acidification of endocytic vesicles) resulted in drastic reduction of transfection efficiency in HeLa cells, which indicated that acidic environment might be necessary for solubilization of carbonate apatite to release DNA from the apatite.

To establish this notion, the present inventors generated fluoridated carbonate apatite to see the effect of solubility of the particles on transfection efficiency, since incorporation of fluoride reduces the solubility of the apatite. The changes in luciferase expression were investigated when the concentration of fluoride ion (from 0.01 to 3 millimolar) or strontium ion (from 0.01 to 3 millimolar) was increased during formation of DNA/carbonate apatite particles. Those results are shown in Fig. 4(B).

Surprisingly, transfection efficiency was reduced gradually to a significant extent (100 fold) with increasing fluoride level in carbonate apatite (Fig. 4(B)).

To rule out the possibility that reduced transfection efficiency of fluoridated carbonate apatite was due to reduced cellular uptake of DNA, the present inventors performed transfection with pEGFP, labeled with PI. Fig. 4(C) illustrates the uptake of Pl-labeled plasmid DNA (pEGFP-N2) and GFP expression for carbonate apatite and fluoridated carbonate apatite, as observed after 20 hours following 4 hours incubation with the particles and treatment with 5 mM EDTA in PBS.

With similar level of intracellular plasmid DNA, while almost 50% of the cells showed GFP expression for carbonate apatite (top row of Fig. 4(C)), no GFP-positive cell was observed for fluoridated carbonate apatite (bottom row of Fig. 4(C)).

To enhance the efficiency of transgene expression for short term uptake of DNA, the present inventors investigated the effect of slightly reducing the solubility of carbonate apatite by low amount of fluoride. Fig. 4(D) illustrates changes in luciferase expression for 1M concentrations of F- added during formation of DNA/carbonate apatite particles. Here, After incubation of cells with the particles, cells were washed with EDTA and grown for 1 day, as described above.

Surprisingly, addition of only 1 iM NaF during generation of carbonate apatite, caused over 15- fold enhancement in luciferase expression and a similar level of efficiency could also be observed for treatment of chloroquine (Fig. 4(D)) known to raise endosomal pH and protect DNA against nuclease.

These observations suggest that an intermediate rate of DNA release during proton consumption and endosome buffering by the low F-containing crystals, could avoid significant nuclease degradation, as also evident from persistent fluorescence of PI (not shown), thus providing more intact DNA for transcription and translation. [5] Study of pH-dependent dissolution behaviors of apatites.

To establish a relationship between transfection efficiency and dissolution rates of the apatites, turbidity measurement was done as an indicator of their solubilization, following an acid load in solution of generated apatites.

Dissolution profiles of different apatites, generated as described in transfection protocol, using 6 mM Ca²⁺ and no DNA, were determined by adjusting the pHs of apatite suspensions to 7.0 and 6.8 with 1 M HCl, followed by turbidity measurements at 320nm at different intervals by V-500 (Jasco, Japan) spectrophotometer. SPS 1500 VR Atomic Absorption Spectrophotometer was used to determine released Ca²⁺ after mild shaking (6 hours at 10 rpm) of lyphilized powder of different apatites in acetate buffer (pH 6.5 and 5.5) at 37°C.

Fig. 5(A) illustrates dissolution rates (at pH 7.0) of fluoridated carbonate apatites prepared by addition of 0-3 mM F- during generation of carbonate apatite at pH 7.5 (described in experimental protocol), were studied by turbidity measurement at 320 nm of apatite suspensions just after being adjusted to the pH 7.0 with 1 N HCl.

Carbonate apatite generated in presence of increasing concentrations of NaF, showed gradual decrease in dissolution rates, as evident from changes in turbidity, following adjustment of pH from 7.5 to 7.0 with 1 N HCl (Fig. 5(A)), which is consistent with gradually reduced transfection efficiency of fluoridated carbonate apatites (Fig. 4(B)).

To examine whether dissolution rates of apatites are correlated with their degree of crystallization, the present inventors studied x-ray diffraction of the apatites (Figs. 1(D), (E) and (F)), which clearly indicates that apatite with higher degree of crystallization, had lower solubility (see Supplementary Fig. 5(A)). In other words, apatites with higher crystallinity (Figs. 1(D) and (H)), showed lower transfection efficiency (Figs. 2(C) and 4(B)).

The gradual increase in crystallinity owing to increased level of incorporated fluoride in carbonate apatite (Figs. 1 (F) to (H)) resulted in gradual decrease in transfection efficiency (Fig. 4(B)). To establish that decreased transfection efficiency was only due to decreased solubility of fluoridated carbonate apatite, but not by any other fluoride-mediated effects, the present inventors examined the effects of strontium which, when incorporated into carbonate apatite, is known to improve the crystallinity and reduce the solubility of the apatite, but to a lesser extent than fluoride. Fig. 5(B) illustrates the solubilities of carbonate apatite, fluoridated carbonate apatite and carbonate apatite containing strontium at pH 7 and pH 6.8.

With decreasing pH from 7.5 to pH 7.0 or pH 6.8 carbonate apatite was completely solubilized within 1 min, whereas fluoridated carbonate apatite was partially dissolved (Fig. 5(B)).

As expected, addition of strontium chloride during preparation of carbonate apatite reduced its dissolution rate but to a level less than that observed for fluoride (Fig. 5(B)). Moreover, transfection efficiency was gradually decreased with increasing concentrations of strontium chloride during generation of DNA/carbonate apatite particles (Fig. 4(B)). Taken together, our findings suggest that intracellular release of DNA through dissolution of apatite should play a major role in carbonate apatitemediated transfection.

When the present inventors compared degree of dissolution of the apatites including hydroxyapatite, based on atomic emission spectroscopic measurement of free Ca²⁺ level, released from the apatite powders in Na-acetate buffer solutions of pH 6.5 and 5.5, fluoridated carbonate apatite was found to dissolve to lower extent than carbonate apatite, and to a higher extent than hydroxyapatite (see Supplementary Fig. 5(C)). To clarify that release of crystal-bound DNA in endosomes is, indeed, a crucial factor for subsequent gene expression, the present inventors allowed PI (a pH-insensitive dye)-labeled plasmid DNA for cellular uptake for 4 hr and removed cell-asssociated DNA with EDTA (Figs. 5(D)-a and -d) and waited for more 5 hr (Figs. 5(D)-b, -c and -e). Fluorescence intensity of PI was significantly quenched after 5 hr due to the nuclease-mediated degradation of free plasmid DNA (Fig. 5(D)-b and -e), but remained sustained for the cells treated with bafilomycin A1 after 4 hr DNA uptake (Fig. 5(D)-c).

DNA was also found almost undegraded (Fig. 5(D)-g) when uptake of DNA was mediated by fluoridated carbonate apatite (Fig. 5(D)-f), indicating that release of DNA is essential for expression in spite of high rate of hydrolysis. In addition to this, the high dissolution rate of carbonate apatite might also contribute to the destabilization of endosomes for DNA release to the cytoplasm (see Supplementary Fig. 3(C)), since v-ATPase-driven massive proton accumulation for crystal dissolution could lead to passive chloride influx to endosomes and subsequent endosome swelling and rupture.

INDUSTRIAL APPLICABILITY

As has been described above, since the transfection agent according to the present invention has high cell transfection efficiency, superior reproducibility and biocompatibility, it can be preferably used in gene recombination techniques, treatment of various diseases and so forth.

Claims

CLAIMS 1. A delivery agent for delivering a target substance to cells comprising; at least compound particles composed of the target substance and a calcium phosphate-based material; wherein, in the case where pH has been changed from pH 8.0 to pH 6.0, the compound particles have a characteristic that at least 50% of the compound particles present at pH 8.0 dissolve within a predetermined time after changing the pH to 6.0.

2. The delivery agent according to claim 1 , wherein the average particle diameter of the compound particles is 500 nanometers or less.

3. The delivery agent according to claim 1 or 2, wherein the calcium phosphate-based material is carbonate apatite.

4. The delivery agent according to any one of claims 1 to 3, wherein the target substance is a negatively charged substance.

5. The delivery agent according to any one of claims 1 to 4, wherein the target substance is at least one type of substance selected from the group consisting of drug, protein and polynucleotide.

6. A method of delivering a target substance to cells comprising: a step of delivering the target substance to the cells by using the delivery agent according to any one of claims 1 to 5.

7. A method for producing a delivery agent used to deliver a target substance to cells that contains compound particles composed of the target substance and carbonate apatite, comprising: a step of forming the compound particles by preparing a composition at least containing calcium ion, phosphate ion, hydrogen carbonate ion and the target substance.

8. The method for producing a delivery agent according to claim 7 comprising: a step of preparing a first solution that contains the calcium ion and the target substance, a step of preparing a second solution that contains the phosphate ion and the hydrogen carbonate ion, and a step of mixing the first solution and the second solution to prepare the composition.

9. The method for producing a delivery agent according to claim 7 or 8, wherein the calcium ion concentration of the composition is 0.1 millimolar or more.

10. The method for producing a delivery agent according to any one of claims 7 to 9, wherein the phosphate ion concentration of the composition is 0.1 millimolar or more.

11. The method for producing a delivery agent according to any one of claims 7 to 10, wherein the hydrogen carbonate ion concentration of the composition is 1.0 millimolar or more.

12. The method for producing a delivery agent according to any one of claims 7 to 11, wherein the composition additionally contains fluoride ion or strontium ion.

13. The method for producing a delivery agent according to any one of claims 7 to 12, wherein the pH of the composition is pH 6.0 to pH 9.0.

14. The method for producing a delivery agent according to any one of claims 7 to 13, wherein the compound particles are formed by holding the composition at 10°C or higher.

15. A composition for producing a delivery agent used to deliver a target substance to cells that contains compound particles composed of the target substance and carbonate apatite; wherein, the composition contains at least calcium ion, phosphate ion and hydrogen carbonate ion, and the delivery agent is produced by adding the target substance to the composition.

16. A composition for producing a delivery agent used to deliver a target substance to cells that contains compound particles composed of the target substance and carbonate apatite; wherein, the composition contains at least phosphate ion and hydrogen carbonate ion, and the delivery agent is produced by adding the target substance and calcium ion to the composition.

17. A kit for producing a delivery agent used to deliver a target substance to cells that contains compound particles composed of the target substance and carbonate apatite; wherein, the kit is composed of: a first component containing at least calcium ion, and a second component containing at least phosphate ion and hydrogen carbonate ion, and the delivery agent containing the compound particles is produced by adding the target substance to the first component followed by mixing the first component and the second component.