JP2012116826A - Titanium dioxide composite, dispersant thereof, and production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a titanium dioxide composite in which an organic compound is made complex with a titanium dioxide-containing carrier which is safe for living body and has satisfactory dispersiblity in a dispersion medium having pH close to neutrality, and the organic compound can be converted into an active type by excitation of the titanium dioxide in the carrier.SOLUTION: The titanium dioxide composite comprises an inactive organic compound composed to a carrier including a titanium dioxide fine particle and a cationic polymer having a biocompatible hydrophilic graft chain, which is ion-bonded to the fine particle surface.

Description

The present invention relates to a composite of titanium dioxide in which a predetermined organic compound is combined with a carrier composed of titanium dioxide fine particles and a polymer that improves the dispersibility of the fine particles. More specifically, when the complex is irradiated with ultraviolet rays or ultrasonic waves, the organic compound can be converted from an inactive form to an active form through excitation of the titanium dioxide fine particles, and administered to a living body. The present invention relates to a titanium dioxide composite.
Moreover, this invention relates to the dispersion liquid and manufacturing method of said titanium dioxide composite.

  Titanium dioxide is known to have a property of generating active oxygen species such as hydroxy radicals and singlet oxygen when irradiated with ultraviolet rays or ultrasonic waves in a dispersion medium. Titanium dioxide itself is also known to have almost no toxicity to living bodies. For this reason, attempts have been made to destroy the cancer cells and tumor tissues in the living body by utilizing the property of generating the reactive oxygen species by administering titanium dioxide fine particles to the living body.

  However, since the isoelectric point of titanium dioxide is around pH 6, there is a problem that titanium dioxide fine particles aggregate and precipitate in a dispersion medium having a pH near neutral. For this reason, it was difficult to administer the titanium dioxide fine particles themselves to a living body.

Various methods have been studied and developed so far in order to improve the dispersibility of the titanium dioxide fine particles near the neutral pH. For example, Japanese Patent No. 3775432 (Patent Document 1) discloses a method of stably dispersing titanium dioxide fine particles in a dispersion medium by esterifying a hydrophilic polymer to the surface of titanium dioxide fine particles. .
Japanese Patent No. 4169078 (Patent Document 2) discloses a method of stably dispersing titanium dioxide fine particles in a dispersion medium by bonding a nonionic water-soluble polymer to the surface of the titanium dioxide fine particles. ing.
Further, in Japanese Patent No. 443677 (Patent Document 3), titanium dioxide fine particles are stabilized in a dispersion medium by binding a hydrophilic polymer amine to the surface of titanium dioxide fine particles and positively charging the surface. A method of dispersing in is disclosed.

  Japanese Patent Application Laid-Open No. 2007-63253 (Patent Document 4) discloses a titanium dioxide composite in which an anticancer agent is bonded to titanium dioxide fine particles to which a hydrophilic polymer as described above is bonded via a functional group of the polymer. Is disclosed. In addition, in this titanium dioxide composite_body | complex, it aims at decomposing | disassembling an anticancer agent by the photoexcitation of titanium dioxide, and lose | disappearing the medicinal effect.

However, although the composite of titanium dioxide fine particles and polymer obtained by the methods described in Patent Documents 1 and 2 shows good dispersibility in a dispersion medium having a pH near neutral, The manufacturing process was complicated.
The composite of titanium dioxide fine particles and polymer amine obtained by the method described in Patent Document 3 also exhibits good dispersibility in a dispersion medium having a pH near neutral. However, since the complex can interact with not only cancer cells but also normal cells due to the positive charge on the surface, there is a concern in terms of safety to living bodies.
The production method of the titanium dioxide composite described in Patent Document 4 is also complicated. In the composite, since the hydrophilic polymer is bonded to the surface of the titanium dioxide fine particles by an ester bond, the polymer may be detached from the titanium dioxide fine particles by excitation of titanium dioxide.

Japanese Patent No. 3775432 Japanese Patent No. 4169078 Japanese Patent No. 4423676 JP 2007-63253 A

  In view of the circumstances as described above, the present invention provides a carrier containing titanium dioxide that is safe for a living body and exhibits good dispersibility even in a dispersion medium having a pH near neutral, and an organic compound is added to the carrier. An object of the present invention is to provide a titanium dioxide composite which can be complexed and convert the organic compound into an active form by excitation of titanium dioxide in a carrier. Another object of the present invention is to provide a dispersion containing the complex and a method for producing the complex.

The present inventor has paid attention to the fact that the surface of titanium dioxide fine particles is negatively charged in a dispersion medium having a pH near neutral. And, by ionic bonding a cationic polymer having a hydrophilic graft chain excellent in biocompatibility on the surface of the titanium dioxide fine particles, it exhibits good dispersibility in a dispersion medium having a pH near neutrality, It was also found that a carrier that can be safely administered to a living body can be obtained.
Furthermore, the present inventor is able to support an inert organic compound on this carrier, and that the organic compound can be converted to an active type by the active oxygen species generated by the excitation of the titanium dioxide fine particles in the carrier. As a result, the present invention has been completed.

That is, according to the present invention, an inert organic compound is combined with a carrier composed of titanium dioxide fine particles and a cationic polymer having a biocompatible hydrophilic graft chain ionically bonded to the surface of the fine particles. When the composite carrier is irradiated with ultraviolet rays or ultrasonic waves, the organic compound can be converted into an active form through excitation of the titanium dioxide fine particles, for administration to a living body A titanium dioxide composite is provided.
Moreover, according to this invention, the dispersion liquid and manufacturing method of said titanium dioxide composite are provided.

  According to the present invention, a titanium dioxide composite which exhibits good dispersibility even in a dispersion medium having a pH near neutrality and has a biocompatible hydrophilic graft chain, can be safely administered to a living body. Can be provided. Further, according to the present invention, it is possible to provide a dispersion of the titanium dioxide composite and a simple method for producing the composite.

2 is a photograph of a dispersion liquid when titanium dioxide fine particles or a carrier used in the titanium dioxide composite of the present invention is dispersed in dispersion media having different pHs. It is a photograph of the dispersion liquid when the carrier used for the titanium dioxide composite of the present invention is dispersed in dispersion media having different salt concentrations. It is the graph which showed the fluorescence intensity of the fluorescein produced | generated from APF when an ultrasonic wave was irradiated to the dispersion liquid of each support | carrier containing aminophenyl fluorescein (APF). It is the graph which showed the survival rate of this cell when each support | carrier is administered to a cultured cell. It is an elution chart when the titanium dioxide composite of the present invention is measured by gel permeation chromatography. It is a photograph of a cell when a titanium dioxide complex (Example 1) is administered to a cultured cell and this is irradiated with ultrasonic waves. It is a photograph of a cell when a titanium dioxide complex (Example 2) is administered to a cultured cell and this is irradiated with ultrasonic waves. It is the graph which showed the survival rate of the cell under each condition when a titanium dioxide complex (Example 3) or a support | carrier (manufacture example 5) is administered to a cultured cell and these are irradiated with an ultrasonic wave.

  In the present specification, the “carrier” means a carrier for carrying an organic compound and administering it to a living body. Such carriers also include carriers used in the art as drug delivery systems (DDS) for delivering a drug only to a target site, such as a tissue, organ or lesion site to be administered.

The carrier that is one of the components of the titanium dioxide composite of the present invention is characterized by comprising titanium dioxide fine particles and a cationic polymer having a biocompatible hydrophilic graft chain that is ionically bonded to the surface of the fine particles. And
In the present specification, the carrier includes not only a carrier in which a cationic polymer having a biocompatible hydrophilic graft chain is ionically bonded to the surface of the titanium dioxide fine particles, but also the titanium dioxide fine particles by the polymer. A carrier whose surface is coated or modified is also included.

  The titanium dioxide fine particles contained in the carrier are not particularly limited as long as they are excited by irradiation with ultraviolet rays or ultrasonic waves in a dispersion medium, that is, generate active oxygen species by irradiation with ultraviolet rays or ultrasonic waves. The crystal form of titanium dioxide may be any of anatase type, rutile type and brookite type.

  The volume average particle diameter of the titanium dioxide fine particles is not particularly limited, but it is 1 to 50 nm, preferably 5 to 20 nm in consideration of accumulation of the titanium dioxide composite of the present invention in a tissue, organ or lesion site. The titanium dioxide fine particles may be a single particle or a mass composed of a plurality of particles.

In the present specification, the “cationic polymer having a biocompatible hydrophilic graft chain” means a graft copolymer obtained by binding a biocompatible hydrophilic graft chain to a cationic polymer.
Further, in this specification, “biocompatible hydrophilic graft chain” (hereinafter, also simply referred to as “graft chain”) is low toxic to living tissues and cells, and immune reactions and thrombi in the living body. It means a graft chain composed of a hydrophilic polymer that does not cause a formation reaction. The biocompatibility of such a hydrophilic polymer can be confirmed by a method or test known per se in the art. Examples of such a method include, after adding the polymer solution to cultured cells, measuring the number of surviving cells, and measuring the production amount of inflammatory cytokines by ELISA. .

  The cationic polymer is not particularly limited as long as it has a plurality of amino groups, but is preferably a water-soluble polymer having a plurality of side chains containing amino groups. Examples of such cationic polymers include polyallylamine, polyethyleneimine, polyvinylamine, polyvinylamidazole, or polyamino acids having a side chain containing a cationic functional group, such as polylysine, polyarginine, and polyhistidine. . Among these, polyallylamine and polylysine are preferable, and polyallylamine is more preferable.

Although the average molecular weight of said cationic polymer is not specifically limited, For example, the number average molecular weight measured by gel permeation chromatography (GPC) is 1,000-100,000, Preferably, it is 5,000-50,000.
In the carrier in the titanium dioxide composite of the present invention, the weight ratio of the cationic polymer to the titanium dioxide fine particles (cationic polymer / titanium dioxide fine particles) is preferably 0.5 to 5, and more preferably 1. ~ 2.

  Examples of the polymer that can be used for the graft chain include (co) polymers having, as monomer units, ethylene glycol, glucose, acrylamide derivatives, or methacrylamide derivatives such as 2-methacryloyloxyethyl phosphorylcholine. Among such (co) polymers, polyethylene glycol, dextran and polymethacryloyloxyethyl phosphorylcholine are preferable, and polyethylene glycol is more preferable.

  The average molecular weight of the graft chain is not particularly limited. For example, the number average molecular weight measured by GPC is 1,000 to 20,000, preferably 2,000 to 10,000.

  The cationic polymer having the graft chain can be obtained, for example, by a method of graft copolymerizing a biocompatible hydrophilic polymer with the cationic polymer. Such a method is not particularly limited and can be appropriately selected from methods known per se in the art. Examples thereof include Grafting-to method, Grafting-from method, Macromonomer method and the like.

The introduction ratio of the biocompatible hydrophilic graft chain to the cationic polymer is usually 1 to 80%, preferably 10 to 40%.
In this specification, “introduction ratio of biocompatible hydrophilic graft chain to cationic polymer” refers to the biocompatible hydrophilic graft chain among the monomer units constituting one molecule of the cationic polymer. It means the percentage of monomer units bonded in percentage.

  When the above graft chain is bonded to the cationic polymer by a covalent bond, one amino group of the cationic polymer is lost. Therefore, in one molecule of the cationic polymer, if the number of graft chains is excessive, ionic bonding with the titanium dioxide fine particles can be affected. Therefore, in the above case, the graft chain introduction ratio to the cationic polymer is preferably 1 to 50%, more preferably 1 to 40%.

The cationic polymer having a graft chain can be ionically bonded to the surface of the negatively charged titanium dioxide fine particles by the positive charge of the amino group. Both ionic bonds can be confirmed by analytical methods known per se in the art.
For example, when an aqueous solution of a cationic polymer having a graft chain containing a high concentration of sodium chloride and an acidic dispersion of titanium dioxide fine particles are mixed, and the pH of the resulting mixture is adjusted to neutral, A method for confirming the occurrence of aggregation and precipitation of titanium dioxide fine particles may be mentioned. In this method, since the formation of ionic bonds is inhibited by ions that are present in large amounts under high salt concentration conditions, it can be confirmed that the above-described polymer and titanium dioxide fine particles were ion-bonded.

In the carrier constituting the titanium dioxide composite of the present invention, the presence of the graft chain makes the surface of the carrier electrically neutral. That is, in the embodiment of the present invention, the surface potential (zeta potential) of the carrier is usually +10 mV or less, preferably +5 mV or less, more preferably +2 mV or less.
The volume average particle diameter of the carrier constituting the titanium dioxide composite of the present invention is not particularly limited, but considering accumulation in cells, tissues, organs or lesion sites, it is usually 10 to 400 nm, preferably 50 to 300 nm. It is.

The titanium dioxide composite of the present invention is characterized in that an inert organic compound is combined with the above carrier. Here, “composite” is not particularly limited as long as it is in a state where an inert organic compound is supported on the carrier.
In the titanium dioxide composite of the present invention, the principle by which the inert organic compound is combined with the carrier is not particularly limited. For example, the compound may be complexed to the polymer layer of the carrier by physical adsorption due to its hydrophobicity, and the compound is retained via the biocompatible hydrophilic graft chain of the carrier. It may be compounded by. In the embodiment of the present invention, it is preferable that the inactive organic compound is combined with the polymer layer of the carrier by physical adsorption due to its hydrophobicity.

  In this specification, for the hydrophobicity of organic compounds, the definition of “solubility” described in “14th revised Japanese Pharmacopoeia Description” (Tokyo Yodogawa Shoten, A-14, General Rules 23 (2001)) is used. refer. That is, in this specification, hydrophobicity (solubility) is defined as the degree to which an organic compound dissolves within 30 minutes when put into water and shaken vigorously for 30 seconds every 20 minutes at 20 ± 5 ° C. Is done. In this definition, the case where the amount of water required to dissolve 1 g of the organic compound is 100 ml or more and less than 1,000 ml is “not easily soluble in water”, and the case where it is 1,000 ml or more and less than 10,000 ml is “very soluble in water” “It is difficult”, and the case of 10,000 ml or more is expressed as “almost insoluble in water”. In the embodiment of the present invention, the inactive organic compound is preferably an organic compound that is hardly soluble in water, extremely hardly soluble, or hardly soluble in water.

  The amount of the inactive organic compound contained in the titanium dioxide composite of the present invention is usually 1 to 300 mg, preferably 5 to 200 mg per 1 g of titanium dioxide, although it varies depending on the type and properties of the compound.

In the present invention, an “inactive organic compound” is reacted with an active oxygen species to change from an inactive form that does not express a predetermined activity in its intact molecular structure to an active form that can express the activity. If it is an organic compound which can convert a molecular structure, it will not specifically limit. In the embodiment of the present invention, the inactive organic compound is preferably an organic compound that is hardly soluble in water, extremely hardly soluble, or hardly soluble in water. In the embodiment of the present invention, the inactive organic compound includes a pharmaceutical compound and a reagent.
As an inactive type organic compound, for example, a modifying group for preventing a predetermined activity from being bonded to the organic compound of the main body is bonded, and the modifying group is eliminated by the action of reactive oxygen species. And compounds capable of expressing the activity. In the art, it is known that reactive oxygen species cleave ester bonds and ether bonds. Therefore, in an inactive organic compound, the above-mentioned modifying group is bonded to the main organic compound via an ester bond or an ether bond. It is preferable that it is couple | bonded with. The modifying group for preventing the predetermined activity of the organic compound from being expressed can be appropriately selected from those known in the art based on the molecular structure and properties of the organic compound of the main body.

When the titanium dioxide composite of the present invention is used as a means of DDS, the inactive organic compound is a molecular structure as it is by reacting with active oxygen species generated by excitation of titanium dioxide fine particles by ultraviolet rays or ultrasonic waves. Then, a pharmaceutical compound whose molecular structure can be converted from an inactive form that does not express a predetermined pharmacological activity in vivo to an active form that can express the pharmacological activity (hereinafter also referred to as “pharmaceutical compound precursor”) Is preferred. More preferably, such pharmaceutical compound precursors are hydrophobic.
In the above-described pharmaceutical compound precursor, a modifying group for preventing a predetermined pharmacological activity from being expressed in vivo is bonded to the pharmaceutical compound of the main body, and the modifying group is eliminated by the action of reactive oxygen species. Therefore, there is no particular limitation as long as it is an organic compound that can express the pharmacological activity. In the pharmaceutical compound precursor, for example, the above-described modifying group is preferably bound to the pharmaceutical compound of the main body via an ester bond or an ether bond. Such pharmaceutical compound precursors may be synthesized by introducing appropriate modifying groups into the pharmaceutical compound of interest by methods known in the art. Commercially available prodrugs can also be used.

  Specific pharmaceutical compound precursors include, for example, carmofur, tegafur, simvastatin, salazosulfapyridine, acyclovir, indomethacin farnesyl, oseltamivir, carisoprodol, enalapril, valacyclovir, phosamprenavir, levodopa, chloramphenicol Acid esters, sirocibin, codeine, molsidomine, paliperidone, primidone, dipivefrin, risdexamphetamine, docosahexaenoic acid paclitaxel, and the like.

The titanium dioxide composite according to the present invention is the above-mentioned inactive organic compound through excitation of titanium dioxide fine particles when the support in which the inactive organic compound is combined is irradiated with ultraviolet rays or ultrasonic waves. Can be converted into an active form.
That is, in the embodiment of the present invention, by irradiating the titanium dioxide composite of the present invention with ultraviolet rays or ultrasonic waves, the titanium dioxide fine particles in the composite are excited to generate active oxygen species. Then, this active oxygen species reacts with the inactive organic compound in the titanium dioxide complex, whereby the compound can be converted into an active form.
Therefore, according to the titanium dioxide composite of the present invention, the activity of the combined organic compound can be adjusted by irradiation with ultraviolet rays or ultrasonic waves from the outside.

In a preferred embodiment of the present invention, when a titanium dioxide complex of the present invention in which a pharmaceutical compound precursor corresponding to the disease is complexed is administered to a patient with a certain disease, ultraviolet rays or ultrasonic waves are applied to a predetermined site of the patient. , The precursor can be converted into the main body pharmaceutical compound at that site. That is, the pharmacological activity of the pharmaceutical compound can be expressed only in a specific tissue, organ or lesion site of the patient.
Therefore, since the titanium dioxide complex of the present invention can express pharmacological activity only at a necessary site, side effects of the above-mentioned pharmaceutical compound can be reduced.

  Whether or not the inactive organic compound in the complex is converted into the active form in the living body administered with the titanium dioxide complex of the present invention can be confirmed by a method known per se in the art. . For example, when the titanium dioxide complex of the present invention is administered to cultured cells, an extract of the cells is prepared after irradiating the cells with ultrasonic waves, and the extract is subjected to high performance liquid chromatography (HPLC). The organic compound converted into the active form can be detected more. Similarly, when the titanium dioxide complex of the present invention is administered to a human, blood is collected from the human after irradiating the specific site of the human, and the blood is converted into an active form by subjecting the blood to HPLC. More organic compounds can be detected.

  The titanium dioxide composite of the present invention is well dispersed in a dispersion medium having a pH near neutral, and is safe for the living body due to the presence of the biocompatible polymer. It can be in the form of a dispersion for uniform administration in a dispersion medium such as an acceptable buffer solution (hereinafter referred to as “dispersion of the present invention”).

  The administration method of the dispersion of the present invention may be any of injection (intravenous, intraarterial, intramuscular, subcutaneous, intradermal, intrathecal, intraperitoneal, etc.), oral, topical application, eye drop, etc. When the above-mentioned pharmaceutical compound precursor is complexed with a carrier, an administration method capable of accumulating the titanium dioxide complex of the present invention at a lesion site in a living body is preferable.

  When the inactive organic compound is a pharmaceutical compound precursor, the dispersion of the present invention can be administered to a living body as it is, but the type of the precursor, the disease to be administered, and the lesion site In consideration of the above, it is also possible to obtain a preparation containing the dispersion of the present invention, which is appropriately selected from various dosage forms. This preparation may further contain a pharmaceutically acceptable additive as long as it does not inhibit the dispersibility of the titanium dioxide composite of the present invention.

The dispersion of the present invention or a preparation containing the dispersion is administered to a living body, and after the titanium dioxide complex of the present invention is accumulated in a tissue, organ or lesion part of the living body, these parts are irradiated with ultraviolet rays or ultrasonic waves. Thus, the active oxygen species can be generated through the titanium dioxide fine particles in the composite, and the inactive organic compound carried can be converted into the active type. In this case, the apparatus or instrument for irradiating ultraviolet rays or ultrasonic waves is not particularly limited as long as it can irradiate ultraviolet rays or ultrasonic waves on the surface and inside of the living body.
When the titanium dioxide composite of the present invention is administered to a human, for example, an ultraviolet lamp or an endoscope equipped with an ultraviolet fiber can be used for ultraviolet irradiation. In addition, for example, an ultrasonic diagnostic apparatus having a probe for locally irradiating ultrasonic waves can be used for ultrasonic irradiation.

  The intensity of ultraviolet rays or ultrasonic waves to be irradiated and the irradiation time are not particularly limited as long as they do not adversely affect the living body and can convert an inactive organic compound into an active form. For example, the wavelength of ultraviolet light is usually 200 to 400 nm, preferably 240 to 280 nm. The frequency of the ultrasonic wave is usually 10 kHz to 3 MHz, preferably 40 kHz to 2 MHz. Moreover, the irradiation time of an ultraviolet-ray or an ultrasonic wave is 5 seconds-10 minutes normally, Preferably it is 10 seconds-5 minutes. In the present invention, since ultrasonic waves have higher biological tissue permeability than ultraviolet rays, it is preferable to irradiate ultrasonic waves.

The production method of the titanium dioxide composite of the present invention (hereinafter referred to as “the production method of the present invention”)
(1) A step of mixing an acidic dispersion of fine titanium dioxide particles and a solution of a cationic polymer having a biocompatible hydrophilic graft chain at pH 1 to 4,
(2) The carrier obtained by adjusting the pH of the mixed solution obtained in the step (1) to 7 to 8 and ion-bonding the titanium dioxide fine particles and the cationic polymer having a biocompatible hydrophilic graft chain. Obtaining
(3) a step of purifying the carrier obtained in the step (2);
(4) including a step of combining the carrier and the inactive organic compound by mixing the carrier purified in the step (3) and the inactive organic compound.
According to the production method of the present invention, the drug delivery carrier of the present invention can be easily obtained.

  The acidic dispersion of titanium dioxide fine particles used in the production method of the present invention is not particularly limited as long as the titanium dioxide fine particles are uniformly dispersed in an acidic dispersion medium. Such an acidic dispersion can be obtained, for example, by heating a titanium tetrachloride aqueous solution or a titanium sulfate aqueous solution and peptizing hydrous titanium dioxide obtained by hydrolysis with an inorganic acid such as nitric acid or hydrochloric acid. . A commercially available acidic titanium dioxide sol may also be used.

  The concentration of the titanium dioxide fine particle acidic dispersion is not particularly limited, but when the volume average particle size of the titanium dioxide fine particles contained in the acidic dispersion is 5 nm, it is usually 1 to 50 mg / ml, preferably 5 to 30 mg / ml. ml.

  The solution of the cationic polymer having a biocompatible hydrophilic graft chain used in the production method of the present invention is a solution obtained by dissolving the polymer in an appropriate aqueous solvent. Such an aqueous solvent is not particularly limited as long as it is an aqueous solvent having a salt concentration that does not hinder the composite (ion bond) of the polymer and titanium dioxide fine particles in the subsequent second step. Examples of such an aqueous solvent include water and physiological saline.

  The concentration of the cationic polymer having a biocompatible hydrophilic graft chain in the solution is not particularly limited, but the weight ratio of the cationic polymer to the titanium dioxide fine particles (cationic polymer / titanium dioxide fine particles) is 0.5 to The concentration may be 5.0, preferably 1.0 to 2.0.

In the first step of the production method of the present invention, the acidic dispersion of the titanium dioxide fine particles and the polymer solution are mixed at pH 2 to 4 to obtain a raw material mixture.
In this step, an acid such as hydrochloric acid or nitric acid may be appropriately added to the above mixed solution before and / or during mixing in order to keep the pH range from 2 to 4. Further, since this step can be performed under normal temperature and normal pressure conditions, it is not necessary to heat and / or pressurize the above-mentioned mixed solution.

  Next, in the second step of the production method of the present invention, the pH of the mixed solution obtained in the first step is adjusted to 7 to 8, and the cationic property having titanium dioxide fine particles and biocompatible hydrophilic graft chains is obtained. The polymer is ionically bonded.

In this step, the pH of the mixed solution is adjusted to 7 to 8 by adding an appropriate amount of an alkali solid or aqueous solution such as sodium hydroxide or potassium hydroxide to the above mixed solution and mixing. Thereby, the surface of the titanium dioxide fine particles in the mixed solution is negatively charged. And the support | carrier which comprises the titanium dioxide composite of this invention is formed when the cationic polymer which has a graft chain combines with this microparticles | fine-particles via an ionic bond.
In addition, since the second step can be performed under conditions of normal temperature and normal pressure, it is not necessary to heat and / or pressurize the above mixed solution before and after pH adjustment.

  In the third step of the production method of the present invention, the carrier obtained in the second step is purified. The purification method is not particularly limited as long as the carrier can be separated from the polymer that has not been complexed with the titanium dioxide fine particles, and examples thereof include dialysis, ultrafiltration, and gel filtration chromatography. .

In the fourth step of the production method of the present invention, the carrier purified in the third step and the inactive organic compound are mixed to form a composite of the carrier and the organic compound. A titanium dioxide composite is obtained.
In this step, it is preferable that the carrier dispersion and the inert organic compound solution are mixed to form a composite of both.
The concentration of titanium dioxide contained in the carrier dispersion is not particularly limited, but is usually 0.01 to 20 mg / ml, preferably 0.1 to 10 mg / ml. The concentration of the inactive organic compound is not particularly limited and can be determined as appropriate. The solvent for dissolving the organic compound is not particularly limited as long as it has a property that does not hinder the above-described complexation. In the present invention, as described above, the inactive organic compound is preferably a hydrophobic organic compound, and therefore the solvent can be appropriately selected from organic solvents such as DMSO and ethanol.

In the fourth step, the ratio between the amount of the inactive organic compound solution and the amount of the carrier dispersion is not particularly limited, but is usually 1: 1 to 100, preferably 1: 2 in volume ratio. 50.
In this step, since the carrier and the organic compound are mixed by diffusion, it is not necessary to perform stirring or the like. However, the speed for stirring is not particularly limited and can be appropriately determined. The mixing time is not particularly limited, but usually 1 to 10 minutes is sufficient.
In the fourth step, it is preferable that the obtained mixed solution is allowed to stand after the mixing of the two ends. The time for standing still is not particularly limited, and 10 minutes or more is sufficient. It can also be left overnight.
In addition, since the fourth step can be performed under conditions of normal temperature and normal pressure, it is not necessary to heat and / or pressurize the mixed solution during mixing and standing.

  After the fourth step, the obtained titanium dioxide composite may be purified as necessary. The purification method is not particularly limited as long as it can remove the inactive organic compound that has not been complexed to the carrier, and examples thereof include a dialysis method, an ultrafiltration method, and a gel filtration chromatography method.

Among the above purification methods, when purified by a dialysis method, an ultrafiltration method or a gel filtration chromatography method, the titanium dioxide complex is uniformly dispersed in water or a buffer solution. By removing the dispersion medium from the dispersion using a method such as freeze drying or reprecipitation, the titanium dioxide composite powder of the present invention can be obtained.
The dispersion of the present invention can be obtained by uniformly dispersing the obtained powder in a dispersion medium that can be administered to a living body, such as water, physiological saline, or a physiologically acceptable buffer.
Furthermore, in the embodiment of the present invention, when the inactive organic compound is a pharmaceutical compound precursor, the titanium dioxide composite powder obtained as described above and a pharmaceutical known in the art are used. It can be set as the pharmaceutical composition containing the titanium dioxide composite_body | complex of this invention by mixing with a pharmaceutically acceptable additive.

  EXAMPLES The present invention will be described in detail below by examples, but the present invention is not limited to these examples.

1. Production of carrier (Production Examples 1 to 4) used in the titanium dioxide composite of the present invention (1) Deprotonation of polyallylamine (PAA) Polyallylamine hydrochloride (1 g) (number average molecular weight 15000: Nittobo Co., Ltd.) Was added to 1M aqueous sodium hydroxide solution (16 ml) and stirred for 3 hours to obtain a PAA solution. Next, the solution was put into a dialysis membrane Spectra / P Membrane (molecular weight fraction 2000: SPECTRUM), and dialyzed in pure water for 24 hours until the pH of the solution became 7-8. Thereafter, the content liquid was recovered from the dialysis membrane and freeze-dried to obtain deprotonated PAA (0.58 g). The ¹ H NMR measurement value of this PAA is shown below.
¹ H NMR (400 MHz, D ₂ O): d 1.22, 1.53 (s, -CH ₂ CH-), 2.61 (s, -CH ₂ NH _2- )

(2) Activation of polyethylene glycol (PEG) Under an argon atmosphere, PEG (30 g) (number average molecular weight 2000: Sigma-Aldrich) was dissolved in THF (400 ml) to obtain a PEG solution. Also, under an argon atmosphere, chloroformate (9.0
g) was dissolved in THF (20 ml) to obtain a chloroformate solution. Triethylamine (9.4 g) was added to the above PEG solution under an argon atmosphere, and the above chloroformate solution was further added dropwise, followed by standing for 3 days. Then, the precipitate was filtered, and the solvent was concentrated from the obtained filtrate by distilling off the solvent under reduced pressure. After adding to chloroform (200 ml), the solution was separated three times using saturated saline (600 ml). went. And the chloroform layer was dripped at diethyl ether (2000 ml), and the deposit was obtained. The precipitate was suction filtered, and the filtrate was dried under vacuum to collect a white solid. This solid was dissolved in benzene (100 ml) and then lyophilized to obtain a white solid (22.0 g) again. The ¹ H NMR measurement value of this solid is shown below.
¹ H NMR (400 MHz, CDCl ₃ ): d 1.79 (s, THF), 3.38 (s, -OCH ₃ ), 3.66 (t, -OCH _2- ), 4.44 (s, -CH ₂ OCO-), 7.40 (t, -CHCNO ₂ ), 8.27 (t, -COOCCH-)

(3) Synthesis of PAA having PEG graft chain (PAA-g-PEG) Lithium chloride (265 mg) was dissolved in methanol (250 ml) to obtain a lithium chloride solution. The PAA (100 mg) obtained in (1) above was dissolved in the solution (110 ml) to obtain a PAA solution. Further, PEG (535 mg or 1070 mg) obtained in (2) above was dissolved in the above lithium chloride solution (110 ml) to obtain PEG solution 1 and PEG solution 2, respectively.

After the PEG solution 1 was added dropwise to the PAA solution, it was allowed to stand for 3 days. This solution was put into a dialysis membrane Spectra / R Membrane (molecular weight fraction 15000; SPECTRUM) and dialyzed against pure water for 48 hours. Thereafter, the content liquid was recovered from the dialysis membrane, and 1 M HCl was added thereto to adjust the pH to 4.5. The solution was passed through a cation exchange column (SP Sepharose; manufactured by Amersham Biosciences), the solvent was evaporated by distillation under reduced pressure, and freeze-dried to obtain a PAA-g-PEG solid (490 mg: (Hereinafter referred to as “PAA-g-PEG1”). Further, in place of the PEG solution 1 described above, a PEG solution 2 was used to obtain a PAA-g-PEG solid (892 mg: hereinafter referred to as “PAA-g-PEG2”). Here, in PAA-g-PEG1 and PAA-g-PEG2, the introduction ratio of the PEG graft chain to PAA was 20% and 40%, respectively.
Moreover, the molecular weight distribution of each obtained PAA-g-PEG was measured by gel permeation chromatography (GPC). As a result, the number average molecular weights of PAA-g-PEG1 and PAA-g-PEG2 were 79000 and 143000, respectively. The ¹ H NMR measurement value of each PAA-g-PEG is shown below.

(PAA-g-PEG1)
¹ H NMR (400 MHz, CD ₃ OD): d 1.18, 1.50 (s, -CH ₂ CH-), 2.57 (s, -CH ₂ NH _2- ),, 3.71 (t, -OCH _2- ), 4.21 (s, -CH ₂ NHCOO-)
(PAA-g-PEG2)
¹ H NMR (400 MHz, CD ₃ OD): d 1.19, 1.38 (s, -CH ₂ CH-), 2.97 (s, -CH ₂ NH _2- ),, 3.71 (t, -OCH _2- ), 4.24 (s, -CH ₂ NHCOO-)

(4) Composite of titanium dioxide fine particles and PAA-g-PEG Titanium dioxide sol STS-100 (2.0 ml, average particle size 5 nm, concentration 25 mg / ml, pH 1.5; Ishihara Sangyo Co., Ltd.) at room temperature and atmospheric pressure And a PAA-g-PEG1 aqueous solution (23.0 ml) with each concentration were mixed to obtain a mixed solution. The concentration of the PAA-g-PEG1 aqueous solution is shown in Table 1 below.
Subsequently, 0.02 M sodium hydroxide aqueous solution (5.0 ml) was dripped at the obtained liquid mixture, and the support | carrier (manufacture example 1 and 2) was obtained by adjusting pH of this liquid mixture to 7-8.

  Instead of the above-mentioned PAA-g-PEG1 aqueous solution, each concentration of PAA-g-PEG2 aqueous solution (23.0 ml) or PAA aqueous solution (23.0 ml) was used, respectively, in the same manner as described above. 3 and 4) and a comparative carrier (Comparative Examples 1 and 2) were obtained. The concentration of each aqueous solution of PAA-g-PEG2 and PAA used is shown in Table 1 below.

  Table 1 shows the weight ratio of PAA to titanium dioxide fine particles and the average particle diameter of each carrier in the obtained carriers. The average particle size of each carrier was measured by a dynamic light scattering method. Also, measure the average particle size of the dispersion of each carrier (2.0 ml) that was freeze-dried and then re-dispersed with water (2.0 ml) or physiological saline (2.0 ml). did. Further, the surface potentials (zeta potentials) of the carriers of Production Examples 2 and 4 were measured and found to be 0.68 mV and 0.93 mV, respectively. That is, it was shown that the surface of these carriers was covered with electrically neutral PEG.

2. Dispersibility of carrier in dispersion medium at neutral pH Pure water was added to the titanium dioxide sol used in step 1 to obtain an aqueous dispersion (pH 2.5) of titanium dioxide fine particles themselves (tube 1 in FIG. 1). When an aqueous sodium hydroxide solution was added to this dispersion to adjust the pH to 7.4, the titanium dioxide fine particles aggregated to form white turbidity (tube 2 in FIG. 1). On the other hand, the above 1. Each of the carriers of Production Examples 1 to 4 and Comparative Examples 1 and 2 obtained in Example 1 was well dispersed even in a dispersion medium having a pH of 7.4 (for example, the carrier dispersion of Production Example 2 was dispersed. , Shown as tube 3 in FIG. 1).
From FIG. 1, it can be seen that only the titanium dioxide fine particles aggregate in a dispersion medium having a pH near neutral, but the carrier obtained by combining the titanium dioxide fine particles with a polymer can be well dispersed in the dispersion medium.

3. Confirmation of ionic bond between titanium dioxide fine particles and cationic polymer having graft chain in carrier In the production of the carrier of Production Example 2 above, as a PAA-g-PEG1 solution mixed with titanium dioxide sol, PAA-g-PEG1 By comparing the case of using an aqueous solution (referred to as Solution A) with the case of using a solution (referred to as Solution B) in which PAA-g-PEG1 is dissolved in 1M aqueous solution of thorium chloride, titanium dioxide fine particles and PAA-g We examined whether or not -PEG was complexed by ionic bonding.
Each solution and titanium dioxide sol were mixed and then allowed to stand for 24 hours. And sodium hydroxide aqueous solution was added to each liquid mixture, and pH was adjusted to 7.4. As a result, when the solution A was used, the carrier was dispersed, but when the solution B was used, the titanium dioxide fine particles aggregated to cause white turbidity. The result is shown in FIG.
This is because ionic interaction between PAA-g-PEG and titanium dioxide fine particles is shielded at high salt concentration. Therefore, it can be seen that in the carrier used in the titanium dioxide composite of the present invention, the titanium dioxide fine particles and PAA-g-PEG are complexed through ionic bonds.

4). Examination of the ability of the carrier to generate reactive oxygen species The ability of the carrier to generate reactive oxygen species was examined using aminophenylfluorescein (APF: manufactured by Sekisui Medical Co., Ltd.). It is known that APF does not fluoresce in its original molecular structure, but reacts with the hydroxyl radical of reactive oxygen species, so that its modifying group is eliminated and converted to fluorescein, increasing its fluorescence intensity. It is a reagent.
A DMF solution of APF (9 μl, 5 mmol / l) was added to the aqueous dispersion of the carrier of Production Example 2 (2991 μl, titanium dioxide concentration 4.0 mg / ml). Then, while stirring the dispersion, after irradiating ultrasonic waves (40 kHz; manufactured by AS ONE, 1 MHz; manufactured by Nepagene) under light-shielding conditions, the dispersion was excited at 485 nm, and the fluorescence at 515 nm was obtained. The strength was measured with a multiplate reader (ARVO SX; manufactured by PerkinElmer). Further, the fluorescence intensity was measured in the same manner using an aqueous dispersion of the carrier of Comparative Example 2 instead of the carrier of Production Example 2. These results are shown in FIG.

  In the aqueous dispersion of the carrier of Production Example 2, an increase in fluorescence intensity depending on the ultrasonic irradiation time was observed, indicating that the carrier can generate reactive oxygen species by ultrasonic irradiation. In addition, since this carrier can generate reactive oxygen species by irradiation with ultrasonic waves of 40 kHz and 1 MHz, it is considered that it can generate reactive oxygen species in a wide frequency range.

5. Examination of Toxicity of Carrier to Cells The presence or absence of toxicity of the carrier obtained above to cells was examined using the human cervical cancer cell line HeLa.
Each carrier of Production Example 2, Production Example 4 and Comparative Example 2 was dispersed in physiological saline to obtain each dispersion having a titanium dioxide concentration of 4.0 mg / ml. HeLa cells are seeded at 5 × 10 ⁴ cells / well in a 24-well plate (manufactured by NUNK), and in DMEM medium (Nissui Pharmaceutical Co., Ltd.) containing 10% fetal calf serum (FBS), 37 ° C., 5% Culturing was performed in a CO ₂ atmosphere. After culturing for 24 hours, the prepared dispersions were added to each well so that the final concentration of titanium dioxide was 0.04 mg / ml, and further cultured for 4 hours. Then, after removing the medium and washing the cells, DMEM medium containing 10% FBS was added and further cultured for 24 hours. Then, the absorbance of each well was measured using MTT Cell Proliferation Assay Kit (Funakoshi Co., Ltd.) according to the instruction manual attached to the kit. The cell viability when each carrier dispersion was added was calculated by comparison with the measured value of the cells to which no dispersion was added. The result is shown in FIG.

FIG. 4 shows that when the carrier dispersion of Comparative Example 2 is added, the cell viability is reduced to 50% or less. That is, this result shows that the carrier of titanium dioxide fine particles complexed only with the cationic polymer PAA is toxic to the living body.
On the other hand, when the dispersions of the carriers of Production Examples 2 and 4 were added, the cell viability was hardly reduced. This is thought to be because the cytotoxicity of PAA was reduced by the PEG graft chain, which is a hydrophilic polymer excellent in biocompatibility.
From these results, it was suggested that the carrier used in the titanium dioxide composite of the present invention can be safely administered to a living body.

6). Production of Titanium Dioxide Complexes of the Present Invention (Examples 1 and 2) 0.5 mM Singlet Oxygene Sensor Green (SOSG: Molecular) was added to the carrier dispersion (200 μl, titanium dioxide concentration 4.0 mg / ml) of Production Example 2 above. Probes) methanol solution (40 μl) was added and allowed to stand overnight to obtain a titanium dioxide composite (Example 1) of the present invention in which SOSG was combined with a carrier. Note that SOSG is usually a reagent that emits weak blue fluorescence, but is known to emit green fluorescence similar to fluorescein by reacting with singlet oxygen.
In addition, in place of the SOSG aqueous solution, a physiological saline (36 μl) was mixed with 5 mM aminophenylfluorescein (APF: Sekisui Medical Co., Ltd.) DMF solution (4 μl) to make 40 μl, A titanium dioxide composite of the present invention (Example 2) obtained by combining APF with a support was obtained.
In these complexes, it was confirmed using GPC measurement that SOSG and APF were supported on the carrier. Specifically, GPC measurement was performed on these dispersions using a TSK Gel PW5000 column. In this measurement, detection was performed by absorption at 300 nm for titanium dioxide, 514 nm for SOSG, and 494 nm for APF. The result is shown in FIG.

As shown in FIG. 5, in the detection chart of titanium dioxide (300 nm) and the detection chart of each reagent (SOSG: 514 nm, APF: 494 nm), each peak was observed in the same elution volume. Normally, when SOSG and APF are separately measured by GPC, no peak is observed in such elution volume.
Therefore, from FIG. 5, it was confirmed that the carriers in the complexes of Examples 1 and 2 carry SOSG and APF, respectively. Since SOSG and APF are hydrophobic reagents with low solubility in water, it is considered that they are supported by being combined with the polymer layer of the carrier.

7). Examination of intracellular delivery of titanium dioxide complex and conversion of organic compound into active form in cells by ultrasonic irradiation Delivering the titanium dioxide complex of Examples 1 and 2 into cells, We examined whether SOSG and APF were converted to active forms emitting green fluorescence by ultrasonic irradiation.
A dispersion (240 μl) of the titanium dioxide composites of Examples 1 and 2 was mixed with a DMEM medium (1760 μl: manufactured by Nissui Pharmaceutical Co., Ltd.) to prepare a mixed solution. A glass bottom dish (manufactured by Matsunami) was seeded with HeLa cells (2 × 10 ⁵ cells) and cultured for 48 hours. And the liquid mixture prepared in this dish was added, and it culture | cultivated for further 4 hours. Thereafter, the cells were irradiated with ultrasonic waves (1 MHz: manufactured by Nepagene) for 10 minutes under light-shielding conditions, and the cells were observed with a laser confocal microscope (manufactured by ZEISS). The results are shown in FIG. 6 and FIG.

  From FIG. 6 and FIG. 7, it was observed that the titanium dioxide complex containing both SOSG and APF was taken into the cells and emitted green fluorescence in the cells by ultrasonic irradiation. This confirms that SOSG and APF were delivered into the cell as a titanium dioxide complex, and that SOSG and APF were converted to active forms that fluoresce via excitation of titanium dioxide fine particles by ultrasonic irradiation. It was.

8). Production of Titanium Dioxide Composite Containing Pharmaceutical Compound Precursor (Example 3) A titanium dioxide composite comprising a pharmaceutical compound precursor and a carrier is produced and administered to a cell, and the cell is irradiated with ultrasonic waves. To examine the efficacy of the pharmaceutical compound. As a pharmaceutical compound precursor, carmofur (1-hexylcarbamoyl-5-fluorouracil), a prodrug of fluorouracil, which is a kind of anticancer agent, was used.
(1) Production of titanium dioxide complex of the present invention containing a prodrug Titanium dioxide sol (1.0 ml, average particle size 5 nm, concentration 2.675 mg / ml, pH 1.5; Ishihara Sangyo Co., Ltd.) at room temperature and atmospheric pressure , PAA-g-PEG1 aqueous solution (4.0 ml, 23.75 ml / ml) was mixed to obtain a mixed solution. A carrier (titanium dioxide concentration: 0.445 mg / ml) was obtained by adding 0.02 M aqueous sodium hydroxide solution dropwise to the obtained mixture to adjust the pH of the mixture to 7-8. Furthermore, the obtained carrier is subjected to ultrafiltration (Ultra Filter Unit USY-20: Toyo Roshi Kaisha, Ltd.), the solvent is replaced with a phosphate buffer and concentrated, and the carrier (Production Example 5: Titanium dioxide concentration) is obtained. 1.78 mg / ml) was obtained.

Carmofur is combined with the carrier by adding the DMSO solution (3.56 μl, 10 mg / ml) of Carmofur (Wako Pure Chemical Industries, Ltd.) to the carrier of Preparation Example 5 (200 μl) and allowing to stand overnight. The titanium dioxide composite of the present invention (Example 3) was obtained.
Carmofur is a compound that is insoluble in an aqueous solvent such as a phosphate buffer, but by using the titanium dioxide composite of the present invention, it was possible to disperse carmofur in the aqueous solvent.

(2) Examination of prodrug efficacy by ultrasonic irradiation to cells administered with titanium dioxide complex The titanium dioxide complex of Example 3 and 10% FBS-containing DMEM medium were mixed at 1: 9 (volume ratio). Thus, a dispersion of the titanium dioxide composite was prepared. HeLa cells (2 × 10 ⁵ cells / well) were seeded in a 6-well plate and cultured for 48 hours. After the culture, the medium was removed from the well, the prepared dispersion (2.0 ml / well) was added, and the mixture was further cultured for 4 hours. Thereafter, the cells were washed twice with a phosphate buffer, 10% FBS-containing DMEM medium (5.0 ml / well) was added, and the cells were irradiated with ultrasound (1 MHz, 0.5 W / cm ² ) for 1 minute. The cells were then cultured for 48 hours, washed twice with phosphate buffer, and then MTT reagent (120 μl / well, 10 mg / mL in PBS: Wako Pure Chemical Industries, Ltd.) and 10% FBS-containing DMEM medium (2.0 ml / well) was added and further cultured for 3 hours. Thereafter, the cells were washed twice with phosphate buffer, and 0.1N HCl-containing isopropanol solution (2.0 ml / well) was added to the wells. Then, the absorbance of each well was measured with a Wallac 1420 ARVO _SX multi-label counter (Perkin Elmer Life Science Japan Co., Ltd.), and the cell viability was calculated from the obtained measured values.
Moreover, it replaced with the titanium dioxide composite of said Example 3, and performed the same operation using the support | carrier of manufacture example 5. FIG. The results are shown in FIG.
From FIG. 8, it was shown that the cell viability is reduced by irradiating the cells administered with the titanium dioxide complex combined with carmofur by sonication compared to the case where the cells were not irradiated.

Claims (7)

An inert organic compound is combined with a carrier composed of titanium dioxide fine particles and a cationic polymer having a biocompatible hydrophilic graft chain ionically bonded to the surface of the fine particles.
When the composite carrier is irradiated with ultraviolet rays or ultrasonic waves, the organic compound can be converted into an active form through excitation of the titanium dioxide fine particles,
A titanium dioxide composite for administration to a living body.   The titanium dioxide composite according to claim 1, wherein the inactive organic compound is an organic compound that is converted to an active type by reacting with active oxygen species generated by excitation of the titanium dioxide fine particles.   The titanium dioxide composite according to claim 1 or 2, wherein the inactive organic compound is a pharmaceutical compound that is converted to an active form by reacting with an active oxygen species generated by excitation of the titanium dioxide fine particles.   The titanium dioxide composite according to any one of claims 1 to 3, wherein the carrier has a volume average particle diameter of 50 to 300 nm.   The titanium dioxide composite according to any one of claims 1 to 4, wherein a surface potential of the composite carrier is +10 mV or less.   The dispersion liquid for administering to a biological body containing the titanium dioxide composite of any one of Claims 1-5. (1) A step of mixing an acidic dispersion of fine titanium dioxide particles and a solution of a cationic polymer having a biocompatible hydrophilic graft chain at pH 1 to 4,
(2) By adjusting the pH of the mixed solution obtained in the step (1) to 7 to 8 and ion-bonding the titanium dioxide fine particles and the cationic polymer having a biocompatible hydrophilic graft chain, Obtaining a carrier;
(3) a step of purifying the carrier obtained in the step (2);
(4) Titanium dioxide for administration to a living body, which comprises the step of combining the carrier and the organic compound by mixing the carrier purified in the step (3) with an inert organic compound. A method for producing a composite.