JP2009091345A - Titanium oxide functional molecule composite particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a titanium oxide functional molecule composite particle having a functional molecule coupled with a titanium oxide surface prepared by making a titanium oxide composite particle dispersed in an aqueous solvent with a water-soluble polymer without degrading the water-soluble polymer. <P>SOLUTION: New function can be imparted without degrading the water-soluble polymer by coupling a titanium oxide surface of a titanium oxide composite particle dispersed in an aqueous solvent with the water-soluble polymer with a functional molecule through at least one functional group selected from the group consisting of a carboxy group, an amino group, a diol group, a salicylic acid group, and a phosphoric acid group. The titanium oxide functional molecule composite particle can be used as a tracer experimental material for confirming a moving state in a human body after administering, and as a medical material for diagnosis and treatment conducted by irradiating ultrasonic waves or light to an affected part. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a titanium oxide functional molecule in which a functional molecule is bonded to the surface of titanium oxide without altering the water-soluble polymer with respect to titanium oxide composite particles dispersed in an aqueous solvent with a water-soluble polymer. It relates to composite particles.

  Titanium oxide is said to have an isoelectric point around pH 6. For this reason, the titanium oxide particles are aggregated in an aqueous solvent near neutrality, and it is extremely difficult to uniformly disperse them. Therefore, various attempts have been made so far to uniformly disperse the titanium oxide particles in the aqueous dispersion medium.

  It is known that PEG (polyethylene glycol) is added as a dispersant to improve the dispersibility of titanium oxide particles in a dispersion medium (Patent Document 1 (JP-A-2-307524) and Patent Document 2). JP 2002-60651 A).

  Alternatively, surface-modified titanium oxide fine particles in which a hydrophilic polymer such as polyacrylic acid is bonded to the titanium oxide fine particles via a carboxyl group are also known (see Patent Document 3 (WO 2004/087577)). This technique is intended for the use of anionic polymers such as polyacrylic acid. A surface charge is imparted by a functional group such as a carboxyl group of the anionic polymer, thereby exhibiting stable dispersibility even in a neutral physiological saline close to the in vivo environment and having a photocatalytic activity function.

  Furthermore, studies have been made to impart functionality to titanium oxide. For example, a molecule having a specific binding ability to a target molecule is immobilized on a carboxyl residue that is not involved in the binding of the hydrophilic polymer to the surface-modified titanium oxide fine particles. A titanium dioxide composite having a function has been proposed (see Patent Document 4 (Patent No. 3835700)). This technique gives a surface charge even when a molecule is fixed by a functional group such as a carboxyl group of an anionic polymer, and exhibits a stable dispersibility. On the other hand, the surface charge provided by the functional group directly contributes to the dispersibility, and the surface charge is reduced by immobilizing the molecule on a residue that is not involved in binding. This places a limit on the amount of molecules to be immobilized.

JP-A-2-307524 JP 2002-60651 A WO2004 / 087577 pamphlet Japanese Patent No. 3835700

In the titanium oxide composite particles dispersed in an aqueous solvent with a water-soluble polymer, the present invention retains high dispersibility without altering the water-soluble polymer, and is bonded to the titanium oxide surface. The purpose of the present invention is to provide a titanium oxide functional molecule composite particle and a dispersion thereof that realizes a new functionality in the vicinity of the titanium oxide surface by molecules.

  The present inventors now have a group of carboxyl groups, amino groups, diol groups, salicylic acid groups, and phosphoric acid groups on the titanium oxide surface of titanium oxide composite particles dispersed in an aqueous solvent with a water-soluble polymer. The present inventors have found that by adding a functional molecule through at least one selected functional group, it is possible to achieve a new functionality without altering the water-soluble polymer.

  That is, according to the titanium oxide functional molecule composite particle of the present invention, high dispersibility by the water-soluble polymer is maintained by bonding the functional molecule to the titanium oxide surface without altering the water-soluble polymer. it can. For this reason, it is possible to design a molecule with a high degree of freedom in binding functional molecules. In addition, by binding the functional molecule to the surface of titanium oxide, the reactivity between the radical species generated by ultrasonic waves or ultraviolet irradiation near the titanium oxide surface and the functional molecule is high. For this reason, the reactivity with the functional molecule and the object having an interaction with the functional molecule can be significantly improved. When the object is a cancer cell, the therapeutic effect of cancer by ultrasonic waves or ultraviolet rays can be remarkably improved. Therefore, the titanium oxide functional molecule composite particles of the present invention can also be used as an ultrasonic cancer treatment promoter for promoting ultrasonic cancer treatment performed by irradiating the affected area with ultrasonic waves.

And the titanium oxide functional molecular composite particles of the present invention are:
Titanium oxide particles,
It comprises a water-soluble polymer bonded to the surface of the titanium oxide particles via at least one functional group selected from the group consisting of a carboxyl group, an amino group, a diol group, a salicylic acid group, and a phosphoric acid group. , Titanium oxide composite particles,
A functional molecule formed by further bonding to the surface of the titanium oxide composite particles;
The functional molecule is
(1) having at least one functional group selected from the group of carboxyl group, amino group, diol group, salicylic acid group, and phosphoric acid group,
(2) a) a saturated or unsaturated chain hydrocarbon group having 6 to 40 carbon atoms, b) a saturated or unsaturated 5- to 6-membered heterocyclic group having or not having a substituent, or c) A compound comprising a saturated or unsaturated 5- or 6-membered cyclic hydrocarbon group with or without a substituent,
It is a titanium oxide functional molecule composite particle bonded to the titanium oxide via the functional group.

  The dispersion according to the present invention comprises titanium oxide functional molecule composite particles and a solvent in which the particles are dispersed.

  According to the present invention, oxidation that has achieved high dispersibility without altering the water-soluble polymer, and has provided new functionality in the vicinity of the titanium oxide surface by the functional molecule bonded to the titanium oxide surface. Titanium functional molecule composite particles and dispersions thereof can be provided.

  The titanium oxide functional molecule composite particles according to the present invention include titanium oxide particles, a water-soluble polymer, and a functional molecule. FIG. 1 shows an example of titanium oxide functional molecular composite particles. As shown in FIG. 1, the titanium oxide functional molecule composite particle is obtained by bonding a water-soluble polymer 2 and a functional molecule 3 to the surface of a titanium oxide particle 1. The bond between the titanium oxide particles 1 and the water-soluble polymer 2 and the functional molecule 3 is via at least one functional group selected from a carboxyl group, an amino group, a diol group, a salicylic acid group, and a phosphoric acid group. It is formed.

  That is, since these functional groups form a strong bond with titanium oxide, dispersibility can be maintained regardless of the high catalytic activity of the titanium oxide particles. Since these functional groups form a strong bond with titanium oxide, it is possible to maintain the bond between the water-soluble polymer and the functional molecule regardless of the high catalytic activity of the titanium oxide particles. In addition, from the viewpoint of safety in the body, the binding form in the present invention may be a binding form in which dispersibility is ensured 24 to 72 hours after administration to the body. A covalent bond is desirable in that the dispersion under physiological conditions is stable, the water-soluble polymer is not released even after irradiation with ultrasonic waves or ultraviolet rays, and damage to normal cells is small.

  A carboxyl group, an amino group, a diol group, a salicylic acid group, and a phosphoric acid group are functional groups such as trifunctional silanol groups that are three-dimensionally condensed with each other and cover the surface of the titanium oxide particles with the polymer. Unlike the case, since the functional groups do not polymerize, it is considered that an exposed portion can be secured on the surface of the titanium oxide particles as shown in FIG. As a result, the catalytic activity of the titanium oxide particles can be sufficiently exhibited while suppressing the deactivation that may occur when the surface is covered with the polymer.

  The water-soluble polymer bonded to the surface of the titanium oxide particles is in a neutral aqueous solvent in which it is difficult to disperse the titanium oxide particles due to the action of electric charge or hydration. Body particles can be highly dispersed. A method for introducing a functional molecule into a water-soluble polymer bonded to the surface of titanium oxide particles is known. In such a case, in order to chemically bond the water-soluble polymer and the functional molecule, the water-soluble polymer needs to contain a highly reactive polar group. The polar group contained in the water-soluble polymer is lost when the functional molecule is bound. This causes a change in the polarity of the water-soluble polymer itself. That is, it is considered that the balance dispersed by the action of charge or hydration of the water-soluble polymer bonded to the surface of the titanium oxide particle changes before and after the functional molecule is bonded. This can only be achieved by well controlling the balance of charge or hydration associated with the alteration of the water-soluble polymer bound to the surface of the titanium oxide particles. On the other hand, the functional molecule bonded to the surface of the titanium oxide particles in the present invention can maintain high dispersibility due to the water-soluble polymer by bonding the water-soluble polymer without altering it. For this reason, it is possible to design a molecule with a high degree of freedom in binding functional molecules without considering dispersibility change caused by alteration of the water-soluble polymer.

  According to the titanium oxide functional molecule composite particle of the present invention, by introducing a hydrophobic or cell recognizable molecule to the titanium oxide surface as the functional molecule, it is high without altering the water-soluble polymer. Dispersibility can be maintained. Furthermore, high interaction between these molecules and cells in the vicinity of the titanium oxide surface can be exhibited. Since cell-recognizing molecules are bound to the surface of the titanium oxide particles, the reaction rate with radical species generated by ultrasonic or ultraviolet irradiation is improved near the surface of the titanium oxide, and the target for killing cancer cells. Thus, the therapeutic effect of cancer by ultrasonic waves or ultraviolet rays can be remarkably improved. Therefore, the titanium oxide functional molecule composite particle of the present invention is selectively taken into the affected area after administration, and then cell recognition for promoting ultrasonic cancer treatment performed by irradiating the affected area with ultrasonic waves. It can also be used as a therapeutic agent for ultrasonic cancer treatment.

  Further, according to the titanium oxide functional molecule composite particle of the present invention, the water-soluble polymer can be altered by introducing a photoresponsive molecule or radioactive substance into the titanium oxide surface as the functional molecule. And high dispersibility can be maintained. Furthermore, these molecules can be labeled directly on the titanium oxide surface. In particular, with regard to radioactive substances, care must be taken from the viewpoint of safety, but the radioactive substance is bound to the surface of titanium oxide to titanium oxide composite particles dispersed in an aqueous solvent with a water-soluble polymer. After that, the labeling of the particles can be performed by a simple and few process by separating and removing the unbound radioactive substance by an appropriate method. For this reason, there are few opportunities for a radioactive substance to spread outside, and it is excellent in terms of safety. Further, by measuring this with an appropriate instrument, particle imaging and quantitative measurement are possible. Therefore, the titanium oxide functional molecule composite particles of the present invention are used for tracer experimental materials for confirming pharmacokinetics after administration into the body, and for diagnosis and treatment performed by irradiating the affected area with ultrasonic waves or light. It can also be used as a medical material.

  Furthermore, according to the titanium oxide functional molecule composite particle of the present invention, by introducing a radical-responsive molecule to the titanium oxide surface as the functional molecule, high dispersion without altering the water-soluble polymer Sex can be maintained. Furthermore, it is possible to improve the reaction rate with radical species generated by irradiation with ultrasonic waves or ultraviolet rays in the vicinity of the titanium oxide surface and to exhibit high reactivity. If the radical-responsive molecule is one that exhibits fluorescence or luminescence due to reaction with radical species, it is possible to measure particles with an appropriate instrument and to measure the amount of radical species generated. . Therefore, the titanium oxide functional molecule composite particle of the present invention can be used as a medical material for diagnosis and treatment performed by irradiating an affected part with ultrasonic waves or light.

  According to a preferred embodiment of the present invention, the water-soluble polymer used in the present invention is at least one selected from the group consisting of a carboxyl group, an amino group, a diol group, a salicylic acid group, and a phosphoric acid group on the surface of the titanium oxide particles. It is preferable that it couple | bonds through the functional group of. As a result, it is possible to firmly bond to the surface of the titanium oxide particle, and the surface of the titanium oxide particle is covered with a polymer by three-dimensional condensation polymerization such as trifunctional silanol groups. Unlike the functional group, the functional groups are not polymerized with each other, so that it is considered that a large number of exposed portions can be secured on the surface of the titanium oxide particles as shown in FIG. As a result, the catalytic activity of the titanium oxide particles can be sufficiently exhibited while suppressing the deactivation that may occur when the surface is covered with the polymer.

  According to a preferred embodiment of the present invention, the water-soluble polymer used in the present invention is not particularly limited as long as the titanium oxide functional molecular composite particles can be dispersed in an aqueous solvent. Water-soluble polymers having water or cationic properties, and those that impart dispersibility by hydration without charge include water-soluble polymers having nonionic properties, and include at least one of these.

According to a preferred embodiment of the present invention, the water-soluble polymer has a weight average molecular weight of 2000 to 100,000. The weight average molecular weight of the water-soluble polymer is a value determined using size exclusion chromatography. By setting the molecular weight within this range, the titanium oxide functional molecular complex is in an aqueous solvent near neutrality where it is difficult to disperse the titanium oxide particles due to the charge or hydration action of the water-soluble polymer. Body particles can be highly dispersed.
A more preferred range is 5000 to 100,000, and even more preferred is 5000 to 40,000.

  According to a preferred embodiment of the present invention, any water-soluble polymer used in the present invention can be used as long as the titanium oxide functional molecule composite particles can be dispersed in an aqueous solvent as an anionic water-soluble polymer. However, examples of those having a plurality of carboxyl groups include carboxymethyl starch, carboxymethyl dextran, carboxymethyl cellulose, polycarboxylic acids, and copolymers (copolymers) having carboxyl group units. Specifically, from the viewpoint of hydrolyzability and solubility of water-soluble polymers, polycarboxylic acids such as polyacrylic acid and polymaleic acid, and copolymers of acrylic acid / maleic acid and acrylic acid / sulfonic acid monomers ( Copolymer) is more preferably used, more preferably polyacrylic acid.

  When polyacrylic acid is used as the water-soluble polymer having anionic property, the weight average molecular weight of polyacrylic acid is preferably 2000 to 100000, more preferably 5000 to 40000, even more preferably from the viewpoint of dispersibility. Is 5000-20000.

  According to a preferred embodiment of the present invention, any water-soluble polymer used in the present invention can be used as long as the titanium oxide functional molecule composite particles can be dispersed in an aqueous solvent as a water-soluble polymer having a cationic property. However, examples of those having a plurality of amino groups include polyamino acids, polypeptides, polyamines, and copolymers (copolymers) having amine units. Specifically, from the viewpoint of hydrolyzability and solubility of the water-soluble polymer, polyamines such as polyethyleneimine, polyvinylamine, and polyallylamine are more preferably used, and polyethyleneimine is more preferable.

  When polyethyleneimine is used as the water-soluble polymer having cationic property, the weight average molecular weight of polyethyleneimine is preferably 2000 to 100,000, more preferably 5000 to 40000, and further preferably 5000 from the viewpoint of dispersibility. ~ 20,000.

  According to a preferred embodiment of the present invention, any water-soluble polymer used in the present invention can be used as long as the titanium oxide functional molecule composite particles can be dispersed in an aqueous solvent as a water-soluble polymer having nonionic properties. However, a polymer having a hydroxyl group and / or a polyoxyalkylene group is preferable. Preferable examples of such water-soluble polymers include polyethylene glycol (PEG), polyvinyl alcohol, polyethylene oxide, dextran or copolymers containing them, more preferably polyethylene glycol (PEG) and dextran, Polyethylene glycol is preferred.

  When polyethylene glycol is used as the water-soluble polymer having nonionic properties, the weight average molecular weight of polyethylene glycol is preferably 2000 to 100,000, more preferably 5000 to 40,000 from the viewpoint of dispersibility.

  According to a preferred embodiment of the present invention, the functional molecule used in the present invention is bonded to the titanium oxide particle surface, and the functional molecule is a carboxyl group, an amino group, a diol group, a salicylic acid group, and a phosphoric acid group. Having at least one functional group selected from the group of

  According to a preferred embodiment of the present invention, the functional molecule used in the present invention is a) a saturated or unsaturated chain hydrocarbon group having 6 to 40 carbon atoms, b) a saturated or unsaturated group having or not having a substituent. It is a compound comprising a saturated 5- to 6-membered heterocyclic group, or c) a saturated or unsaturated 5- to 6-membered cyclic hydrocarbon group with or without a substituent.

  The functional molecule having the above carbon number has a smaller molecular size than the water-soluble polymer. The functional molecule is bonded to the titanium oxide surface. For this reason, in the titanium oxide functional molecule composite particle of the present invention, the water-soluble polymer is located in the outer shell, whereas it has a structure having the functional molecule in the inner position. The outer shell has the greatest influence on the dispersibility of the titanium oxide functional molecular composite particles. That is, the water-soluble polymer located in the outer shell has less influence on the dispersibility of the functional molecule located inside, and can be suitably used.

  In addition, since the functional molecule consisting of the above carbon number is bonded to the surface of the titanium oxide particle, the reactivity between the radical species generated by ultrasonic wave or ultraviolet irradiation near the titanium oxide surface and the functional molecule. Therefore, the reactivity of the functional molecule and the object having an interaction with the functional molecule can be remarkably improved, and it can be suitably used.

  According to a preferred embodiment of the present invention, the functional molecule used in the present invention is not particularly limited in its function, but is hydrophobic, cell recognizable, photoresponsive, radioactive, radical responsive, pharmaceutical, antibody, etc. And linkers with other biomolecules and other functional molecules.

The amount of the functional molecule bonded to the titanium oxide functional molecule composite particles is 1.0 × 10 ^{−1 to} 1.0 × 10 ⁻⁵ parts by mass / titanium oxide particles per mass of the titanium oxide particles. It is a mass part, It is preferable that it is preferably a mass part of 1.0 * 10 ^<-2 > -1.0 * 10 ^{< -5} > mass part / titanium oxide particle. Within this range, the titanium oxide functional molecule composite particles can be suitably used because they can be dispersed using a 10% protein solution close to the in vivo environment as a solvent.

  Examples of such functional molecules include aromatic compounds and molecules having an alkyl structure, and more specifically, molecules having a benzene ring include catechol, methyl catechol, tertiary butyl catechol dopa, dopamine. And catechol having a catechol structure in the molecule, such as dihydroxyphenylethanol, dihydroxyphenylpropionic acid, and dihydroxyphenylacetic acid. As other cyclic molecules, ferrocene, ferrocene carboxylic acid, ascorbic acid, dihydroxycyclobutene diene, alizarin, binaphthalenediol and the like can be suitably used. Furthermore, examples of the molecule having an alkyl structure include molecules having an alkyl group such as a hexyl group, an octyl group, a lauryl group, a palmityl group, and a stearyl group. Alternatively, an alkenyl group such as a hexenyl group, an octenyl group, and an oleyl group, or a saturated or unsaturated aliphatic hydrocarbon group such as a cycloalkyl group can be used.

  Moreover, as another example of a functional molecule, a peptide or amino acid sequence containing a binding active site may be used in cell recognition. More specifically, 5-aminolevulinic acid, methionine, cysteine, glycine and the like can be mentioned. Alternatively, a sugar chain may be included. Furthermore, it may contain a nucleic acid having binding properties. The nucleic acid is not particularly limited, and in addition to DNA and RNA, nucleobases such as aptamers and peptide nucleic acids such as PNA can also be used.

  In addition, as another example of the functional molecule, a molecule containing a fluorescent molecule can be used in photoresponsiveness.

Further, other examples of the functional molecules, in radioactive include compounds containing isotopes, such as ^{14 C-labeled} catechol with ^{14 C-is} preferably used.

  Other examples of functional molecules include chemiluminescent molecules, fluorescent molecules, or spin trapping agents that exhibit specific reactivity with radicals in radical responsiveness. More specifically, chemiluminescent molecules and fluorescent molecules include luminol, sea firefly luciferin analog, oxalate ester, acridinium, parahydroxyphenylfluorescein, paraaminophenylfluorescein, dihydrorhodamine 123, dihydrorhodamine 6G, trans-1 -(2'-methoxyvinyl) pyrene, dihydroxyethidium, folic acid, (2 ', 7'-dichlorodihydrofluorescein diacetate, succinimidyl ester, Invitrogen), 5-or6- (N-Succinimidyloxycarbonyl) -3', 6'-O'- , O'-diacetylfluorescein, Cy dye (Amersham Biosciences), pterin, etc. Examples of the pin trap agent include 4,6-Tri-tert-butylnitrosobenzene, 2-Methyl-2-nitrosopropane, 3,3,5,5-tetramethyl-1-pyrroline N-Oxide, 5,5-Dimethyl-1-pyrroline. N-Oxide, 5- (Diethylphosphono) -5-methyl-1-pyrroline N-Oxide, N-tert-Butyl-alpha- (4-pyridyl-1-oxide) nitrone, N-tert-Butyl-alpha-phenylene Nitrosorbene, 5,5-Dimethyl-1-pyrroline N-oxide, 4-Hydroxy-2,2 6,6-tetramethylperidinyloxy, free radical, 2- (5,5-Dimethyl-2-oxo-2λ5- [1,3,2] dioxaphosphin-2-yl) -2-methyl-3,4-dihydro-2H- pyrrole 1-oxide, 5-Diethylphosphophoryl-5-methyl-1-pyrroline-N-oxide, and the like.

  Other examples of functional molecules include fluorouracil, gemcitabine, methotrexate, cyclophosphamide, daunorubicin hydrochloride, adriamycin, idarubicin hydrochloride, bleomycin, mitomycin, actinomycin, vincristine, cisplatin when anticancer agents are used. , Carboplatin, etoposide, nedaplatin, paclitaxel, docetaxel, irinotecan hydrochloride and the like. Also, antibacterial agents such as penicillin, macrolide, new quinolone, tetracycline, and other antiviral agents such as lamivudine, nelfinavir, indinavir, saquinavir, interferon, amantadine, acyclovir, and new prorelin, buserelin, goserelin, triptorelin And hormonal disease therapeutic agents such as nafarelin and analgesics such as ibuprofen.

  According to a preferred embodiment of the present invention, there is no problem even if the functional molecule used in the present invention is a molecule in which a molecule that provides the above function and a functional group that binds to the surface of titanium oxide are bonded via a linker. .

  According to a preferred aspect of the present invention, the linker may be, for example, a heterobifunctional crosslinker used when biomolecules are bonded with different functional groups. Specific examples of the linker include N-hydroxysuccinimide, N- [α-maleimidoacetoxy] succinimide ester, N- [β-maleimidopropyloxy] succinimide ester, N-β-maleimidopropionic acid, N- [β-maleimidopropion Acid] hydrazide.TFA, 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride, N- [epsilon] -maleimidocaproic acid, N-[[epsilon] -maleimidocaproic acid] hydrazide, N-[[epsilon] -maleimidocaproyl Oxy] succinimide ester, N- [γ-maleimidobutyryloxy] succinimide ester, N-κ-maleimidoundecanoic acid, N- [κ-maleimidoundecanoic acid] hydrazide, succinimidyl-4- [N-maleimidomethyl] -cyclohexane- -Carboxy- [6-amidocaproate], succinimidyl 6- [3- (2-pyridyldithio) -propionamido] hexanoate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, 4- [4-N-maleimidophenyl] Butyric acid hydrazide.HCl, 3- [2-pyridyldithio] propionylhydrazide, N- [p-maleimidophenyl] isocyanate, N-succinimidyl [4-azidophenyl] -1,3′-dithiopropionate, N-succinimidyl S -Acetylthioacetate, N-succinimidyl S-acetylthiopropionate, succinimidyl 3- [bromoacetamido] propionate, N-succinimidyl iodoacetate, N-succinimidyl [4-iodoacetyl] ami Nobenzoate, succinimidyl 4- [N-maleimidomethyl] -cyclohexane-1-carboxylate, succinimidyl 4- [p-maleimidophenyl] butyrate, succinimidyl 6-[(β-maleimidopropionamido) hexanonate], 4-succinimid Diloxycarbonyl-methyl-α [2-pyridyldithio] toluene, N-succinimidyl 3- [2-pyridyldithio] propionate, N- [ε-maleimidocaproyloxy] sulfosuccinimide ester, N- [γ-maleimidobutyryl Oxy] sulfosuccinimide ester, N- [κ-maleimidoundecanoyloxy] -sulfosuccinimide ester, sulfosuccinimidyl-6- [α-methyl-α- (2-pyridyldithio) toluamide] hexano , Sulfosuccinimidyl 6- [3 ′-(2-pyridylthiothio) -propionamide] hexanonate, m-maleimidobenzoyl-N-hydroxysulfo-succinimide ester, sulfosuccinimidyl [4-iodoacetyl] amino Benzoate, sulfosuccinimidyl 4- [N-maleimidomethyl] -cyclohexane-1-carboxylate, sulfosuccinimidyl 4- [p-maleimidophenyl] butyrate, N- [ε-trifluoroacetylcaproyloxy] Examples include succinimide ester, chlorotriazine, dichlorotriazine, and trichlorotriazine. Further, the linker may be composed of a plurality of types of linkers such that other linkers are bonded to each other.

  According to a preferred embodiment of the present invention, the diol group used for bonding the titanium oxide particles to the water-soluble polymer and / or the functional molecule is preferably an enediol group, more preferably an α-diol group. is there. By using these functional groups, excellent bonding to titanium oxide particles can be realized.

  According to a preferred embodiment of the present invention, the titanium oxide particles are preferably anatase type titanium oxide or rutile type titanium oxide. Anatase-type titanium oxide is preferred when utilizing catalytic activity by irradiation with ultraviolet rays or ultrasonic waves, and rutile-type titanium oxide is preferred when utilizing properties such as a high refractive index as in cosmetics.

  According to the preferable aspect of this invention, the titanium oxide functional molecule composite particle used for this invention has a particle diameter of 20-200 nm, More preferably, it is 50-200 nm, More preferably, it is 50-150 nm. Within this particle size range, when administered to the body of a patient for the purpose of reaching a cancer tumor, the cancer tissue is efficiently reached by Enhanced Permeability and Retention Effect (EPR effect) like a drug delivery system. Accumulated. And as above-mentioned, the specific production | generation of a radical seed | species occurs by irradiation of a 400kHz-20MHz ultrasonic wave and an ultraviolet-ray. Therefore, cancer tissue can be killed with high efficiency by irradiation with ultrasonic waves or ultraviolet rays.

  According to another preferred embodiment of the present invention, when the titanium oxide functional molecule composite particles have a particle diameter of less than 50 nm (for example, several nm), the apparent size can be increased to obtain the EPR effect. That is, a high cancer treatment effect is realized by the EPR effect by being bonded by a method such as connecting semiconductor particles with a polyfunctional linker so as to have a form of a secondary particle having a particle diameter of 50 to 150 nm. be able to. According to still another preferred embodiment of the present invention, in order to utilize the EPR effect, titanium oxide functional molecule complex particles can be included in a drug inclusion body such as a liposome.

  In the present invention, the particle diameter of the semiconductor particles can be measured by a dynamic light scattering method. Specifically, it can be obtained as a value represented by Z-average size obtained by cumulant analysis using a particle size distribution measuring apparatus (Zeta Sizer Nano, manufactured by Malvern Instruments).

  The titanium oxide functional molecular composite particles usable in the present invention include not only a single type of titanium oxide composite particles but also a mixture or composite of a plurality of types of semiconductor particles. Specific examples include a composite of titanium oxide particles and iron oxide nanoparticles, a composite of titanium oxide particles and platinum, and titanium oxide coated with silica.

  According to a preferred embodiment of the present invention, the titanium oxide functional molecule composite particles are preferably dispersed in a solvent to be in the form of a dispersion. Thereby, a titanium oxide functional molecule composite particle can be efficiently administered in a patient's body by various methods, such as drip, injection, and application. The liquid property of the dispersion liquid is not limited, and high dispersibility can be realized over a wide range of pH 3 to 10. From the viewpoint of safety in in vivo administration, the dispersion preferably has a pH of 5 to 9, more preferably 5 to 8, and particularly preferably neutral liquidity. According to a preferred embodiment of the present invention, the solvent is preferably an aqueous solvent, more preferably a pH buffer solution or physiological saline. A preferable salt concentration of the aqueous solvent is 2 M or less, and 200 mM or less is more preferable from the viewpoint of safety in in vivo administration. The titanium oxide functional molecule composite particles are preferably contained in an amount of 0.001 to 1% by mass or less, more preferably 0.001 to 0.1% by mass with respect to the dispersion. Within this range, particles can be effectively accumulated in the affected area (tumor) 24 to 72 hours after administration. That is, the concentration of particles tends to accumulate in the affected area (tumor), and the dispersibility of the particles in the blood is ensured to make it difficult to form a coagulation fistula, resulting in secondary adverse effects such as occlusion of blood vessels after administration. There is no fear.

  The titanium oxide functional molecule composite particles of the present invention can be administered into a patient's body by various methods such as infusion, injection, and application. In particular, when it is used by intravenous or subcutaneous administration route, it is preferable from the viewpoint of reducing the burden on the patient by so-called DDS treatment using the EPR effect due to the size of the particles and the retention in the blood. And the titanium oxide functional molecule composite particle administered in the body reaches the cancer tissue and accumulates like a drug delivery system.

  The titanium oxide functional molecule composite particles of the present invention are irradiated with ultrasonic waves or ultraviolet rays, and can become a cytotoxin by the irradiation. This titanium oxide functional molecule composite particle is administered into the body, is subjected to ultrasonic irradiation, and becomes a cytotoxin by the irradiation, so that cells can be killed, but not only in the body but also in a test tube Cells that are killed can be killed. In the present invention, the subject to be killed is not particularly limited, but is preferably a cancer cell. That is, according to the titanium oxide functional molecule composite particle of the present invention, it can be activated by irradiation with ultrasonic waves or ultraviolet rays to kill cancer cells.

  According to a preferred embodiment of the present invention, ultrasonic treatment is performed on the cancer tissue in which the titanium oxide functional molecule composite particles are accumulated. The frequency of the ultrasonic wave to be used is preferably 400 kHz to 20 MHz, more preferably 600 kHz to 10 MHz, and further preferably 1 MHz to 10 MHz. The ultrasonic irradiation time should be appropriately determined in consideration of the position and size of the cancer tissue to be treated, and is not particularly limited. Thus, the cancer tissue of the patient can be killed with high efficiency by ultrasonic waves, and a high cancer treatment effect can be realized. Ultrasound can reach the deep part in the living body from the outside, and when used in combination with the titanium oxide functional molecule composite particles of the present invention, the affected part that exists in the deep part of the living body in a non-invasive state Treatment of the target site can be realized. Furthermore, when the titanium oxide composite particles of the present invention accumulate on the affected part or target site, the local concentration of the titanium oxide composite particles of the present invention accumulated with weak ultrasonic waves that do not adversely affect the surrounding normal cells. Can only work.

  By the way, the effect that these semiconductor particles are activated by the irradiation of ultrasonic waves to kill the cells can be obtained by generating radical species by the irradiation of ultrasonic waves. That is, it is considered that the biological killing effect given by these semiconductor particles is due to the qualitative and quantitative increase of radical species. The reason is presumed as follows. However, the following reason is a hypothesis, and the present invention is not limited to the following description. That is, hydrogen peroxide and hydroxyl radicals are generated in the system only by ultrasonic irradiation, but according to the knowledge of the present inventors, hydrogen peroxide and hydroxyl radicals are not present in the presence of semiconductor particles such as titanium oxide. Generation is promoted. In addition, in the presence of these semiconductor particles, particularly in the presence of titanium oxide, it seems that the production of superoxide anion and singlet oxygen is promoted. The specific generation of these radical species, when using nanometer order fine particles, is remarkable when the frequency during ultrasonic irradiation is in the range of 400 kHz to 20 MHz, preferably in the range of 600 kHz to 10 MHz, more preferably in the range of 1 MHz to 10 MHz. It is considered that this phenomenon is observed.

  Examples are shown below. Unless otherwise specified, “%” means mass%.

Example 1: Preparation of polyethylene glycol-bonded titanium oxide fine particles 3.6 g of titanium tetraisopropoxide and 3.6 g of isopropanol were mixed, and hydrolyzed by adding dropwise to 60 ml of ultrapure water under ice cooling. After dropping, the mixture was stirred at room temperature for 30 minutes. After stirring, 1 ml of 12N nitric acid was added dropwise, and the mixture was stirred at 80 ° C. for 8 hours for peptization. After completion of the peptization, the solution was filtered with a 0.45 μm filter, and the solution was exchanged using a desalting column PD-10 (Amersham Biosciences) to prepare an acidic titanium oxide sol having a solid content of 1%. This titanium oxide sol was placed in a 100 ml vial and subjected to ultrasonic treatment at 200 kHz for 30 minutes using an ultrasonic generator MIDSONIC 200 (manufactured by Kaijo Corporation). The average dispersed particle diameter after sonication was measured by a dynamic light scattering method. In this measurement, the titanium oxide sol after sonication was diluted 1000 times with 12N nitric acid, and then 0.1 ml of the dispersion was charged into a quartz measurement cell, and a zeta sizer nano ZS (manufactured by Sysmex Corporation) was used. The various parameters of the solvent were set to the same values as in water and carried out at 25 ° C. As a result, the dispersed particle diameter was 20.2 nm. Using an evaporating dish, the titanium oxide sol solution was concentrated at 50 ° C. to finally prepare an acidic titanium oxide sol having a solid component of 20%.

  Next, 5 ml of water was added to 1 g of a polyoxyethylene-monoallyl-monomethyl ether / maleic anhydride copolymer (average molecular weight; 33659—manufactured by Nippon Oil & Fats) and the resulting solution and 1-ethyl-3- (3-Dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Dojindo) was mixed with ultrapure water so that the concentrations were 50 mg / ml and 50 mM, respectively. 4-Aminosalicylic acid (molecular weight Mn = 153.14: MP Biomedicals, Inc.) was mixed with the prepared solution to a concentration of 50 mM to obtain 4 ml of solution. This solution was allowed to react with shaking at room temperature for 72 hours. After the reaction, the resulting solution was transferred to a dialysis membrane Spectra / pore CE dialysis tube (fraction molecular weight = 3500, Spectrum Laboratories, Inc.) and dialyzed against 4 l of ultrapure water at room temperature for 24 hours. went. After dialysis, the whole was transferred to an eggplant flask and freeze-dried overnight, and 4 ml of dimethylformamide (DMF: Wako Pure Chemical Industries, Ltd.) was added to the obtained powder and mixed to obtain a 4-aminosalicylic acid-bonded polyethylene glycol solution.

  Next, using DMF, the 4-aminosalicylic acid-bonded polyethylene glycol solution is adjusted so that the final concentration is 20 (vol / vol)%, and the previously obtained anatase-type titanium dioxide sol has a final concentration of 0.25% solid component. The reaction solution was 2.5 ml. The reaction solution was transferred to a hydrothermal reaction vessel HU-50 (manufactured by Sanai Kagaku) and subjected to a heating reaction at 80 ° C. for 6 hours. After completion of the reaction, the reaction vessel was cooled to a temperature of 50 ° C. or lower, and after removing DMF with an evaporator, 1 ml of distilled water was added to obtain a dispersion of polyethylene glycol-bonded titanium dioxide fine particles as titanium oxide composite particles. . Further, HPLC: AKTA purifier (manufactured by GE Healthcare Bioscience), column: HiPrep 16/60 Sephacryl S-300HR (manufactured by GE Healthcare Bioscience), mobile phase: phosphate buffer solution (pH 7.4), flow rate: 0.3 ml / min], a peak of UV absorption was confirmed in the flow-through fraction, and this fraction was collected. This dispersion was diluted with distilled water to a 0.05 (wt / vol)% aqueous solution and allowed to stand for 72 hours, and then the dispersed particle size and zeta potential were confirmed by dynamic light scattering using zeta sizer nano ZS. A zeta potential measurement cell was charged with 0.75 ml of a dispersion of polyethylene glycol-bonded titanium dioxide fine particles, and various parameters of the solvent were set to the same values as in water and measured at 25 ° C. As a result of cumulant analysis, the dispersed particle size was 32.7 nm.

Example 2: Preparation of polyacrylic acid-bonded titanium oxide fine particles 3.6 g of titanium tetraisopropoxide and 3.6 g of isopropanol were mixed, and hydrolyzed by adding dropwise to 60 ml of ultrapure water under ice cooling. After dropping, the mixture was stirred at room temperature for 30 minutes. After stirring, 1 ml of 12N nitric acid was added dropwise, and the mixture was stirred at 80 ° C. for 8 hours for peptization. After completion of the peptization, the solution was filtered with a 0.45 μm filter, and the solution was exchanged using a desalting column PD-10 (Amersham Biosciences) to prepare an acidic titanium oxide sol having a solid content of 1%. This titanium oxide sol was placed in a 100 ml vial and sonicated at 200 kHz for 30 minutes. The average dispersed particle diameter after sonication was measured by a dynamic light scattering method. In this measurement, the titanium oxide sol after ultrasonic treatment was diluted 1000 times with 12N nitric acid, and then 0.1 ml of the dispersion was charged into the quartz measurement cell using Zeta Sizer Nano ZS (manufactured by Sysmex Corporation). The various parameters of the solvent were set to the same values as in water and carried out at 25 ° C. As a result, the dispersed particle diameter was 20.2 nm. Using an evaporating dish, the titanium oxide sol solution was concentrated at 50 ° C. to finally prepare an acidic titanium oxide sol having a solid component of 20%.

  0.6 ml of this acidic titanium oxide sol was adjusted and dispersed with dimethylformamide (DMF) to 20 ml, and after adding 10 ml of DMF in which 0.3 g of polyacrylic acid having an average molecular weight of 5000 (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved, stirring was performed. And mixed. The solution was transferred to HU-50 (manufactured by Sanai Kagaku Co., Ltd.), a hydrothermal reactor, and reacted at 150 ° C. for 5 hours. After completion of the reaction, the reaction solution was cooled until the reaction vessel temperature was 50 ° C. or lower, and twice the amount of isopropanol was added to the reaction solution. After standing at room temperature for 30 minutes, the precipitate was collected by centrifugation at 2000 g for 15 min. The recovered precipitated surface was washed with ethanol, and 1.5 ml of water was added to obtain a dispersion of polyacrylic acid-bonded titanium oxide fine particles as titanium oxide composite particles. This dispersion was diluted 100 times with distilled water, and the dispersed particle size and zeta potential were measured by a dynamic light scattering method. This measurement was performed at 25 ° C. using Zeta Sizer Nano ZS, charging 0.75 ml of polyacrylic acid-bonded titanium oxide fine particle dispersion into a zeta potential measurement cell, and setting various parameters of the solvent to the same values as water. It was. As a result, the dispersed particle size was 53.6 nm, and the zeta potential was −45.08 mV.

Example 3 Production of Polyethyleneimine Bonded Titanium Oxide Fine Particles 3.6 g of titanium tetraisopropoxide and 3.6 g of isopropanol were mixed and hydrolyzed by adding dropwise to 60 ml of ultrapure water under ice cooling. After dropping, the mixture was stirred at room temperature for 30 minutes. After stirring, 1 ml of 12N nitric acid was added dropwise, and the mixture was stirred at 80 ° C. for 8 hours for peptization. After completion of peptization, the solution was filtered with a 0.45 μm filter, and the solution was exchanged using a desalting column PD-10 (Amersham Pharmacia Bioscience) to prepare an acidic titanium oxide sol having a solid content of 1%. This titanium oxide sol was placed in a 100 ml vial and sonicated at 200 kHz for 30 minutes. The average dispersed particle diameter after sonication was measured by a dynamic light scattering method. In this measurement, after diluting 1000 times with 12N nitric acid, using Zetasizer Nano ZS (manufactured by Sysmex Corporation), 0.1 ml of the dispersion is charged into a quartz measurement cell, and various parameters of the solvent are set to the same values as water. And performed at 25 ° C. As a result, the dispersed particle diameter was 20.2 nm.

  3 ml of the obtained titanium oxide sol was dispersed in 20 ml of dimethylformamide (DMF), and 10 ml of DMF in which 450 mg of polyethyleneimine having an average molecular weight of 10,000 (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved was added and stirred and mixed. The solution was transferred to HU-50 (manufactured by Sanai Kagaku Co., Ltd.), a hydrothermal reactor, and reacted at 150 ° C. for 5 hours. After completion of the reaction, the reaction solution was cooled until the reaction vessel temperature became 50 ° C. or lower, and twice the amount of acetone was added to the reaction solution. After standing at room temperature for 30 minutes, the precipitate was collected by centrifugation at 2000 g for 15 min. The recovered precipitated surface was washed with ethanol, and 1.5 ml of water was added to obtain a dispersion of polyethyleneimine-bonded titanium oxide fine particles as titanium oxide composite particles. This dispersion was diluted 100 times with distilled water, and the dispersed particle size and zeta potential were measured by a dynamic light scattering method. This measurement was performed at 25 ° C. using a Zeta Sizer Nano ZS, in which a 0.75 ml dispersion of polyethyleneimine-bound titanium oxide fine particles was charged in a zeta potential measurement cell, and various parameters of the solvent were set to the same values as water. . As a result, the dispersed particle size was 57.5 nm, and the zeta potential was 47.5 mV.

Example 4: Binding of catechol to titanium oxide composite particles Each titanium oxide composite particle obtained in Examples 1 to 3 was made 1% solid component with ultrapure water. Next, catechol was adjusted with ultrapure water to a molar concentration of 100 mM, 10 mM, 1 mM, and 0.1 mM. Equal amounts of each catechol solution are mixed with 1% solution of titanium oxide composite particles, and finally the titanium oxide composite particles and the catechol solution are finished with phosphate buffered saline (PBS; pH 7.4). The concentration was adjusted to 10-fold dilution. Each of the adjusted solutions was transferred to a thermostat and set to 40 ° C., and a binding reaction was performed for 3 hours. When the absorption spectrum of the wavelength in the visible light region of the solution after the reaction was confirmed by an ultraviolet-visible light spectrophotometer, an increase was observed for each solution, and thus it was considered that catechol was bound. Further, when the solution before and after the reaction was subjected to capillary electrophoresis under the following conditions, the amount of change in catechol was determined by confirming a peak at an absorption wavelength of 214 nm with a photodiode array detector.
・ Device: P / ACE MDQ (manufactured by Beckman Coulter)
Capillary: fused silica capillary 50 μm i. d × 67cm (effective length 50cm) (manufactured by Beckman Coulter)
Mobile phase: 50 mM sodium acetate buffer solution (pH 4.8)
・ Voltage: 25kV
・ Temperature: 20 ℃
From the obtained change amount, the catechol was 5.0 × 10 ^{−3 to} 1.0 × 10 ⁻⁴ catechol-g / titanium oxide particles-g per mass of the titanium oxide particles.
Furthermore, unreacted catechol was removed by recovering 2.5 ml of this solution with 3.5 ml of water using a natural fall column PD-10 (manufactured by GE Healthcare Bioscience) for buffer exchange. The removal of catechol was confirmed by capillary electrophoresis as described above, and it was confirmed that there was no free catechol. From these, production of catechol-bonded titanium oxide functional molecule composite particles was confirmed.

Example 5: Binding of ¹⁴ C-labeled catechol to titanium oxide composite particles In Example 4, ¹⁴ C-labeled catechol (final concentration of ¹⁴ C-labeled catechol 1 mM) was used instead of catechol, and titanium oxide composite particles were obtained in Example 1. ¹⁴ C-labeled catechol binding to titanium oxide composite particles was performed in exactly the same procedure except that only polyethylene glycol-bonded titanium oxide fine particles were used. As a ^result, it was confirmed the binding of ¹⁴ C-labeled catechol, the removal of free ¹⁴ C-labeled catechol. In addition, after each solution was diluted to 0.01% solid component using ultrapure water, the average dispersed particle size was measured in the same manner as in Example 1 using Zeta Sizer Nano ZS (manufactured by Sysmex Corporation). It was. As a result, the dispersed particle size was 35.7 nm. From these, production of ¹⁴ C-labeled catechol-bonded titanium oxide functional molecule composite particles was confirmed.

Example 6: Dispersibility of catechol-bonded titanium oxide functional molecular composite particles The catechol-bonded titanium oxide functional molecular composite particles using the polyethylene glycol-bonded titanium oxide fine particles obtained in Example 4 were each RPMI 1640 containing 10% serum. It added so that it might become 0.01% of solid component with respect to a culture medium (product made from GIBCO), and it left still at room temperature for 96 hours. Thereafter, the average dispersed particle size was measured in the same manner as in Example 1 using Zetasizer Nano ZS. The results are shown in Table 1. From Table 1, it was confirmed that each catechol-bonded titanium oxide functional molecule composite particle has a particle size in the range of 20 to 200 nm.

Example 7: Hydrophobicity of catechol-bonded titanium oxide functional molecular composite particles The dispersions of titanium oxide composite particles and catechol-bonded titanium oxide functional molecular composite particles obtained in Examples 1 to 4 were stirred well. And mixed. After 1 minute, when the state of bubbles on the liquid surface of each solution was visually confirmed, the dispersion of catechol-bonded titanium oxide functional molecular composite particles formed bubbles compared to the dispersion of titanium oxide composite particles. It was confirmed that the catechol-bonded titanium oxide functional molecule composite particles had increased hydrophobicity due to the catechol bond.

Example 8: Preparation of polyethylene glycol-bonded titanium oxide fine particles 3.6 g of titanium tetraisopropoxide and 3.6 g of isopropanol were mixed and dropped into 60 ml of ultrapure water under ice cooling for hydrolysis. After dropping, the mixture was stirred at room temperature for 30 minutes. After stirring, 1 ml of 12N nitric acid was added dropwise, and the mixture was stirred at 80 ° C. for 8 hours for peptization. After completion of the peptization, the solution was filtered with a 0.45 μm filter, and the solution was exchanged using a desalting column PD-10 (manufactured by GE Healthcare Bioscience) to prepare an acidic titanium oxide sol having a solid content of 1%. This acidic titanium oxide sol was placed in a 100 ml vial and subjected to ultrasonic treatment at 200 kHz for 30 minutes using an ultrasonic generator MIDSONIC 200 (manufactured by Kaijo Corporation). The average dispersed particle diameter after sonication was measured by a dynamic light scattering method. In this measurement, the acidic titanium oxide sol after sonication was diluted 1000 times with 12N nitric acid, and then 0.1 ml of the dispersion was charged into a quartz measurement cell, and Zeta Sizer Nano ZS (manufactured by Sysmex Corporation) was used. Then, various parameters of the solvent were set to the same value as water, and the measurement was performed at 25 ° C. As a result, the dispersed particle size was 38.4 nm. Using an evaporating dish, the acidic titanium oxide sol solution was concentrated at 50 ° C. to finally prepare an acidic titanium oxide sol having a solid component of 20%.

  Next, 4.5 ml of ultrapure water was added to 0.5 g of a polyoxyethylene-monoallyl-monomethyl ether / maleic anhydride copolymer (average molecular weight 15000; AM1510K—manufactured by Nippon Oil & Fats) to obtain a polyethylene glycol solution. Next, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; manufactured by Dojindo Chemical Co., Ltd.) was prepared to a concentration of 400 mM using ultrapure water to prepare an EDC solution. 0.25 ml of ultrapure water, 0.1 ml of 0.75N aqueous sodium hydroxide solution, and 0.5 ml of EDC solution were mixed and prepared in 1 ml of polyethylene glycol solution. To the prepared solution, dopamine hydrochloride (molecular weight Mn = 153.178: manufactured by Wako Pure Chemical Industries, Ltd.) was mixed to a concentration of 15 mM to obtain 2 ml of solution. This solution was reacted by shaking for 2 hours at 40 ° C. After the reaction, the resulting solution was transferred to a dialysis membrane Spectra / pore CE dialysis tube (fraction molecular weight = 3500, Spectrum Laboratories, Inc.) and dialyzed against 4 l of ultrapure water at room temperature for 24 hours. went. After dialysis, the whole was transferred to an eggplant flask and freeze-dried overnight. To the obtained powder, 4 ml of dimethylformamide (DMF: Wako Pure Chemical Industries) was added and mixed to obtain a dopamine-bonded polyethylene glycol solution.

  Next, using DMF, a dopamine-bonded polyethylene glycol solution is prepared so that the final concentration is 20 (vol / vol)%, and the previously obtained acidic titanium oxide sol has a final concentration of 0.25% solid component. It was. This reaction solution was transferred to HU-50 (manufactured by Sanai Kagaku), a hydrothermal reaction vessel, and subjected to a heating reaction at 150 ° C. for 6 hours. After completion of the reaction, the reaction vessel was cooled to a temperature of 50 ° C. or lower, and after removing DMF with an evaporator, 1.25 ml of distilled water was added to obtain a dispersion of polyethylene glycol-bonded titanium oxide fine particles. This dispersion is diluted with distilled water to a 0.05 (wt / vol)% aqueous solution and allowed to stand for 24 hours, and then the dispersion particle size and zeta potential are confirmed by dynamic light scattering using zeta sizer nano ZS. A zeta potential measurement cell was charged with 0.75 ml of a dispersion of polyethylene glycol-bonded titanium oxide fine particles, various parameters of the solvent were set to the same values as water, and measurement was performed at 25 ° C. As a result of cumulant analysis, the dispersed particle size was 57.3 nm, and the zeta potential was 3.41 mV.

Example 9: Preparation of polyethylene glycol-bonded titanium oxide fine particles 3.6 g of titanium tetraisopropoxide and 3.6 g of isopropanol were mixed and dropped into 60 ml of ultrapure water under ice cooling for hydrolysis. After dropping, the mixture was stirred at room temperature for 30 minutes. After stirring, 1 ml of 12N nitric acid was added dropwise, and the mixture was stirred at 80 ° C. for 8 hours for peptization. After completion of the peptization, the solution was filtered with a 0.45 μm filter, and the solution was exchanged using a desalting column PD-10 (manufactured by GE Healthcare Bioscience) to prepare an acidic titanium oxide sol having a solid content of 1%. This acidic titanium oxide sol was placed in a 100 ml vial and subjected to ultrasonic treatment at 200 kHz for 30 minutes using an ultrasonic generator MIDSONIC 200 (manufactured by Kaijo Corporation). The average dispersed particle diameter after sonication was measured by a dynamic light scattering method. In this measurement, the acidic titanium oxide sol after sonication was diluted 1000 times with 12N nitric acid, and then 0.1 ml of the dispersion was charged into a quartz measurement cell, and Zeta Sizer Nano ZS (manufactured by Sysmex Corporation) was used. Then, various parameters of the solvent were set to the same value as water and the measurement was performed at 25 ° C. As a result, the dispersed particle size was 38.4 nm. Using an evaporating dish, the acidic titanium oxide sol solution was concentrated at 50 ° C. to finally prepare an acidic titanium oxide sol having a solid component of 20%.

  Next, 4.5 ml of ultrapure water was added to 0.5 g of a polyoxyethylene-monoallyl-monomethyl ether / maleic anhydride copolymer (average molecular weight 30000; AM2090P—manufactured by NOF Corporation) to obtain a polyethylene glycol solution. Next, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; manufactured by Dojindo Chemical Co., Ltd.) was prepared to a concentration of 400 mM using ultrapure water to prepare an EDC solution. 0.25 ml of ultrapure water, 0.1 ml of 0.75N aqueous sodium hydroxide solution, and 0.5 ml of EDC solution were mixed and prepared in 1 ml of polyethylene glycol solution. To the prepared solution, dopamine hydrochloride (molecular weight Mn = 153.178: manufactured by Wako Pure Chemical Industries, Ltd.) was mixed to a concentration of 15 mM to obtain 2 ml of solution. This solution was reacted by shaking for 2 hours at 40 ° C. After the reaction, the resulting solution was transferred to a dialysis membrane Spectra / pore CE dialysis tube (fraction molecular weight = 3500, Spectrum Laboratories, Inc.) and dialyzed against 4 l of ultrapure water at room temperature for 24 hours. went. After dialysis, the whole was transferred to an eggplant flask and freeze-dried overnight. To the obtained powder, 4 ml of dimethylformamide (DMF: Wako Pure Chemical Industries) was added and mixed to obtain a dopamine-bonded polyethylene glycol solution.

  Next, using DMF, the dopamine-bonded polyethylene glycol solution was prepared so that the final concentration was 20 (vol / vol)%, and the acidic titanium oxide sol obtained in Example 1 was 0.25% solid component at the final concentration. A reaction solution was obtained. This reaction solution was transferred to HU-50 (manufactured by Sanai Kagaku), a hydrothermal reaction vessel, and subjected to a heating reaction at 150 ° C. for 6 hours. After completion of the reaction, the reaction vessel was cooled to a temperature of 50 ° C. or lower, and after removing DMF with an evaporator, 1.25 ml of distilled water was added to obtain a dispersion of polyethylene glycol-bonded titanium oxide fine particles. This dispersion is diluted with distilled water to a 0.05 (wt / vol)% aqueous solution and allowed to stand for 24 hours, and then the dispersion particle size and zeta potential are confirmed by dynamic light scattering using zeta sizer nano ZS. A zeta potential measurement cell was charged with 0.75 ml of a dispersion of polyethylene glycol-bonded titanium oxide fine particles, various parameters of the solvent were set to the same values as water, and measurement was performed at 25 ° C. As a result of cumulant analysis, the dispersed particle size was 60.3 nm, and the zeta potential was 3.05 mV.

Example 10: Preparation of polyethylene glycol-bonded titanium oxide fine particles 3.6 g of titanium tetraisopropoxide and 3.6 g of isopropanol were mixed and dropped into 60 ml of ultrapure water under ice cooling for hydrolysis. After dropping, the mixture was stirred at room temperature for 30 minutes. After stirring, 1 ml of 12N nitric acid was added dropwise, and the mixture was stirred at 80 ° C. for 8 hours for peptization. After completion of the peptization, the solution was filtered with a 0.45 μm filter, and the solution was exchanged using a desalting column PD-10 (manufactured by GE Healthcare Bioscience) to prepare an acidic titanium oxide sol having a solid content of 1%. This acidic titanium oxide sol was placed in a 100 ml vial and subjected to ultrasonic treatment at 200 kHz for 30 minutes using an ultrasonic generator MIDSONIC 200 (manufactured by Kaijo Corporation). The average dispersed particle diameter after sonication was measured by a dynamic light scattering method. In this measurement, the acidic titanium oxide sol after sonication was diluted 1000 times with 12N nitric acid, and then 0.1 ml of the dispersion was charged into a quartz measurement cell, and Zeta Sizer Nano ZS (manufactured by Sysmex Corporation) was used. Then, various parameters of the solvent were set to the same value as water, and the measurement was performed at 25 ° C. As a result, the dispersed particle size was 38.4 nm. Using an evaporating dish, the acidic titanium oxide sol solution was concentrated at 50 ° C. to finally prepare an acidic titanium oxide sol having a solid component of 20%.

  Next, 4.5 ml of ultrapure water was added to 0.5 g of 2,4-bis (O-methoxypolyethyleneglycol) -6-chloro-s-triazine (average molecular weight 10,000; manufactured by PEG2-Seikagaku Corporation) and a polyethylene glycol solution. did. 2 ml of 50 mM 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid buffer (pH 7.0) and dopamine hydrochloride (molecular weight Mn = 153.178: Wako Pure Chemical Industries, Ltd.) in 4 ml of polyethylene glycol solution 8 ml of solution was obtained by mixing to a concentration of 2.5 mM. This solution was allowed to react with shaking at room temperature for 24 hours. After the reaction, the resulting solution was transferred to a dialysis membrane Spectra / pore CE dialysis tube (fraction molecular weight = 3500, Spectrum Laboratories, Inc.) and dialyzed against 4 l of ultrapure water at room temperature for 24 hours. went. After dialysis, the whole was transferred to an eggplant flask and freeze-dried overnight. To the obtained powder, 4 ml of dimethylformamide (DMF: Wako Pure Chemical Industries) was added and mixed to obtain a dopamine-bonded polyethylene glycol solution.

  Next, using DMF, the dopamine-bonded polyethylene glycol solution was prepared so that the final concentration was 40 (vol / vol)%, and the acidic titanium oxide sol obtained in Example 1 was 0.25% solid component at the final concentration. A reaction solution was obtained. This reaction solution was transferred to HU-50 (manufactured by Sanai Kagaku), a hydrothermal reaction vessel, and subjected to a heating reaction at 150 ° C. for 6 hours. After completion of the reaction, the reaction vessel was cooled to a temperature of 50 ° C. or lower, and after removing DMF with an evaporator, 1.25 ml of distilled water was added to obtain a dispersion of polyethylene glycol-bonded titanium oxide fine particles. This dispersion is diluted with distilled water to a 0.05 (wt / vol)% aqueous solution and allowed to stand for 24 hours, and then the dispersion particle size and zeta potential are confirmed by dynamic light scattering using zeta sizer nano ZS. A zeta potential measurement cell was charged with 0.75 ml of a dispersion of polyethylene glycol-bonded titanium oxide fine particles, various parameters of the solvent were set to the same values as water, and measurement was performed at 25 ° C. As a result of cumulant analysis, the dispersed particle size was 123 nm, and the zeta potential was 13.1 mV.

Example 11: Binding of fluorescent molecules to titanium oxide composite particles The titanium oxide composite particles obtained in Example 1 were made into a dispersion of 1% solid component with ultrapure water. Next, dopamine hydrochloride (molecular weight Mn = 153.178: manufactured by Wako Pure Chemical Industries, Ltd.) was adjusted to 200 mM. The prepared solution and dispersion were mixed at 1: 9 to make 1 ml, and a binding reaction was performed at room temperature for 1 hour. When the absorption spectrum of the wavelength in the visible light region of the solution after the reaction was confirmed by an ultraviolet-visible light spectrophotometer, an increase was observed for each solution, so it was considered that dopamine was bound. Further, when the solution before and after the reaction was subjected to capillary electrophoresis under the following conditions, the amount of change in dopamine was determined by confirming a peak at an absorption wavelength of 214 nm with a photodiode array detector.
・ Device: P / ACE MDQ (manufactured by Beckman Coulter)
Capillary: fused silica capillary 50 μm i. d × 67cm (effective length 50cm) (manufactured by Beckman Coulter)
Mobile phase: 50 mM sodium acetate buffer solution (pH 4.8)
・ Voltage: 25kV
・ Temperature: 20 ℃
From the obtained amount of change, dopamine was 5.2 × 10 ⁻² dopamine-g / titanium oxide particles-g per mass of titanium oxide particles.
Furthermore, unreacted dopamine was removed by recovering 2.5 ml of this solution with 3.5 ml of water using a buffer-replacement natural falling column PD-10 (manufactured by GE Healthcare Bioscience). The removal of dopamine was confirmed by capillary electrophoresis as described above, and it was confirmed that there was no free dopamine. From these, production of dopamine-bound titanium oxide functional molecule composite particles was confirmed.

  Next, the dopamine-bound titanium oxide functional molecule composite particles were mixed and adjusted in a 20 mM borate buffer so that the final concentration was 0.3% and NHS-Rhodamine (Pierce) was 1 mM. This solution was allowed to stand at 4 ° C. under light shielding for 24 hours. Unreacted NHS-Rhodamine was removed from 2.5 ml of this solution using 3.5 ml of water by using a natural drop-type column for buffer exchange PD-10 (manufactured by GE Healthcare Bioscience). The removal of NHS-Rhodamine was confirmed by capillary electrophoresis as described above, and it was confirmed that there was no free NHS-Rhodamine. The obtained solution was subjected to spectrum analysis with a fluorescence spectrophotometer, and it was confirmed that the excitation wavelength was 555 nm and the fluorescence wavelength was 575 nm. From these facts, production of fluorescent molecule-bound titanium oxide functional molecule composite particles in which fluorescent molecules were bound to titanium oxide composite particles was confirmed.

Example 12: Binding of dopamine to titanium oxide composite particles The titanium oxide composite particles obtained in Example 1 were made into a dispersion of 1% solid component with ultrapure water. Next, dopamine hydrochloride (molecular weight Mn = 153.178: manufactured by Wako Pure Chemical Industries, Ltd.) was adjusted to 200 mM. The prepared solution and dispersion were mixed at 1: 9 to make 1 ml, and a binding reaction was performed at room temperature for 1 hour. When the absorption spectrum of the wavelength in the visible light region of the solution after the reaction was confirmed by an ultraviolet-visible light spectrophotometer, an increase was observed for each solution, so it was considered that dopamine was bound. Further, when the solution before and after the reaction was subjected to capillary electrophoresis under the following conditions, the amount of change in dopamine was determined by confirming a peak at an absorption wavelength of 214 nm with a photodiode array detector.
・ Device: P / ACE MDQ (manufactured by Beckman Coulter)
Capillary: fused silica capillary 50 μm i. d × 67cm (effective length 50cm) (manufactured by Beckman Coulter)
Mobile phase: 50 mM sodium acetate buffer solution (pH 4.8)
・ Voltage: 25kV
・ Temperature: 20 ℃
From the obtained amount of change, dopamine was 5.2 × 10 ⁻² dopamine-g / titanium oxide particles-g per mass of titanium oxide particles.
Furthermore, unreacted dopamine was removed by recovering 2.5 ml of this solution with 3.5 ml of water using a buffer-replacement natural falling column PD-10 (manufactured by GE Healthcare Bioscience). The removal of dopamine was confirmed by capillary electrophoresis as described above, and it was confirmed that there was no free dopamine. From these, production of dopamine-bound titanium oxide functional molecule composite particles was confirmed.

Example 13: Binding of dihydroxyphenylpropionic acid to titanium oxide composite particles The titanium oxide composite particles obtained in Example 1 were made into 3% solid component with ultrapure water. Next, dihydroxyphenylpropionic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was adjusted with ultrapure water to a molar concentration of 500 mM. The 1% amount of dihydroxyphenylpropionic acid solution was mixed with the 3% solution of titanium oxide composite particles to prepare 1 ml in total. The prepared solution was subjected to a binding reaction at room temperature for 2.5 hours. When the absorption spectrum of the wavelength in the visible light region of the solution after the reaction was confirmed with an ultraviolet-visible light spectrophotometer, an increase in absorbance was observed, and it was considered that dihydroxyphenylpropionic acid was bound. Further, the amount of change in dihydroxyphenylpropionic acid was determined by confirming the peak at an absorption wavelength of 214 nm of the photodiode array detector under the following conditions by capillary electrophoresis for the solution before and after the reaction.
・ Device: P / ACE MDQ (manufactured by Beckman Coulter)
Capillary: fused silica capillary 50 μm i. d × 67cm (effective length 50cm) (manufactured by Beckman Coulter)
-Mobile phase: 50 mM borate buffer solution (pH 9.0)
・ Voltage: 25kV
・ Temperature: 20 ℃
The dihydroxyphenylpropionic acid bonded from the obtained amount of change was 4.8 × 10 ⁻² dihydroxyphenylpropionic acid-g / titanium oxide-g per mass of titanium oxide particles. Furthermore, unreacted dihydroxyphenylpropionic acid was removed from the 2.5 ml of this solution using 3.5 ml of water by using a buffer-exchanged natural fall column PD-10 (manufactured by GE Healthcare Bioscience). Removal of dihydroxyphenylpropionic acid was confirmed by capillary electrophoresis as described above, and it was confirmed that there was no free dihydroxyphenylpropionic acid. From these, production of dihydroxyphenylpropionic acid-bonded titanium oxide functional molecule composite particles was confirmed.

Example 14: Binding of ethanolamine to dihydroxyphenylpropionic acid-bonded titanium oxide functional molecule composite particles The solution of dihydroxyphenylpropionic acid-bonded titanium oxide functional molecular composite particles obtained in Example 9 and 1-ethyl-3 -(3-Dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Dojindo) was mixed and adjusted using a 50 mM borate buffer solution (pH 9.0) to a concentration of 15 mg / ml and 80 mM, respectively. Ethanolamine (manufactured by Wako Pure Chemical Industries, Ltd.) was mixed with the prepared solution to a concentration of 10 mM to obtain 0.2 ml of solution. This solution was allowed to react with shaking at room temperature for 24 hours. 50 μl of the reacted solution was adjusted to 1000-fold dilution with ultrapure water, and this was added 1/10 to 50 mM borate buffer to make 440 μl. Further, 20 μl of 0.3 mg / ml fluorescamine (manufactured by Tokyo Chemical Industry Co., Ltd.) dissolved in acetone was added and the solution was stirred. Then, the fluorescence value was measured with a fluorometer (manufactured by Shimadzu Corporation: RF-5300) as follows. Measured under conditions. (Wavelength: Ex = 395 nm / Em = 495 nm, Bandwidth: Ex = 5 nm / Em = 10 nm, Sensitivity: High, Response: Auto). Using a calibration curve prepared in advance, unbound ethanolamine was calculated to calculate the rate of concentration change from the initial concentration, that is, the rate of binding. As a result, it was 63.16%. From the above, it was confirmed that ethanolamine was bound via dihydroxyphenylpropionic acid in dihydroxyphenylpropionic acid-bonded titanium oxide functional molecule composite particles.

Example 15: Binding of antibody to dihydroxyphenylpropionic acid-bonded titanium oxide functional molecule composite particles The solution of dihydroxyphenylpropionic acid-bonded titanium oxide functional molecular composite particles obtained in Example 13 and 1-ethyl-3- (3-Dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Dojindo) was mixed with ultrapure water so that the concentrations were 20 mg / ml and 80 mM, respectively. The mixed solution was reacted at room temperature for 10 minutes. The solution was exchanged with a 20 mM HEPES buffer solution (pH 7.4) using a desalting column PD-10 (manufactured by GE Healthcare Bioscience) to obtain a solution of particles having a titanium oxide concentration of 20 mg / ml. An anti-human serum albumin (anti-HSA) monoclonal antibody (mouse IgG: MSU-304, Cosmo Bio) prepared with the same buffer was added to 3 mg / ml to make a total volume of 1 ml. After the reaction at 4 ° C. for 24 hours, ethanolamine was added so that the final concentration was 0.5 M, and the reaction was further performed at 4 ° C. for 1 hour. This solution was adjusted to a titanium oxide concentration of 1 mg / ml, HPLC: AKTA purifier (manufactured by GE Healthcare Bioscience), column: HiPrep 16/60 Sephacryl S-500HR (manufactured by GE Healthcare Bioscience), mobile phase: When 1 ml of phosphate buffered saline (pH 7.4), flow rate: 0.3 ml / min] was applied, the fraction that passed through and the anti-HSA monoclonal antibody used for binding alone were confirmed to absorb UV. Peaks were confirmed and these fractions were collected. The pass-through fraction was diluted with distilled water to a 0.05 (wt / vol)% aqueous solution and allowed to stand for 24 hours, and then the dispersed particle size and zeta potential were confirmed by dynamic light scattering using a zeta sizer nano ZS. A zeta potential measurement cell was charged with 0.75 ml of a dispersion of polyethylene glycol-bonded titanium oxide fine particles, various parameters of the solvent were set to the same value as water, and measurement was performed at 25 ° C. As a result of cumulant analysis, the dispersed particle size was 50.3 nm, and the zeta potential was −3.0 mV. As for the fraction in which the anti-HSA monoclonal antibody was confirmed alone, the protein concentration was measured by the Bradford method, and as a result, a decrease in the antibody concentration was confirmed before and after the reaction. From the above, it was confirmed that the antibody molecule was bound via dihydroxyphenylpropionic acid in the dihydroxyphenylpropionic acid-bonded titanium oxide functional molecule composite particle.

Example 16: Binding of aminolevulinic acid and dihydroxyphenylethanol to titanium oxide composite particles 300 mg of 3,4-dihydroxyphenylethanol (Wako Pure Chemical Industries) was dissolved in 0.5 ml of dimethylformamide (DMF; Wako Pure Chemical Industries). did. To this solution, 0.1 ml of thionyl chloride (manufactured by Wako Pure Chemical Industries) was added and stirred, and then 7 ml of toluene (manufactured by Wako Pure Chemical Industries) was mixed. Furthermore, 250 mg of 5-aminolevulinic acid hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted at 80 ° C. for 1 hour with stirring. After the reaction, an upper layer part and a lower layer part separated into two layers were collected. A portion of this recovered solution and a DMF solution of 3,4-dihydroxyphenylethanol and 5-aminolevulinic acid hydrochloride were subjected to thin layer chromatography (TLC), using acetone (manufactured by Wako Pure Chemical Industries): methanol (Wako Pure Chemical Industries). The product was developed using a solution of 2: 3, and then sprayed with a ninhydrin spray (Wako Pure Chemical Industries) on a dried TLC plate, followed by heating and spot observation. As a result, the developed spot in the lower layer of the recovered liquid was located at a position different from 3,4-dihydroxyphenylethanol and 5-aminolevulinic acid hydrochloride and colored by the ninhydrin reaction. And a complex of 5-aminolevulinic acid hydrochloride. The lower layer portion of the collected liquid was concentrated 10 times under reduced pressure to obtain a reaction concentrated solution. The titanium oxide composite particles obtained in Example 1 were adjusted to 1% solid components with ultrapure water, and 1/10 amount of the reaction concentrated solution was mixed therewith to make 1 ml in total. The mixed solution was reacted at room temperature for 1 hour while gently stirring. After the reaction, the precipitated component was centrifuged (1500 g, 10 min) to collect the supernatant, and 1 ml of this solution was subjected to water 1. using a natural drop column NAP-10 (manufactured by GE Healthcare Bioscience) for buffer exchange. Collected in 5 ml, unreacted 3,4-dihydroxyphenylethanol and 5-aminolevulinic acid hydrochloride complex and DMF removed. Since the absorption spectrum of the wavelength in the visible light region (400 nm) of this solution was confirmed by an ultraviolet-visible light spectrophotometer (UV1600; manufactured by Shimadzu Corporation), an increase was observed, and 3,4-dihydroxyphenylethanol and 5-aminolevulin The hydrochloride complex was thought to be bound. From these, production of complex-bound titanium oxide functional molecular complex particles of 3,4-dihydroxyphenylethanol and 5-aminolevulinic acid hydrochloride was confirmed.

Example 17: Binding of folic acid and dopamine to titanium oxide composite particles Folic acid (manufactured by Wako Pure Chemical Industries, Ltd.) and dopamine hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) were added to dimethylformamide (DMF; manufactured by Wako Pure Chemical Industries, Ltd.) to 1 mM. Dissolved. Similarly, using DMF, 200 mM Benzotriazole-1-yl-oxy-trispyrrolophosphonophosphate phosphophosphate (PyBop; manufactured by Merck), 200 mM 1-hydroxybenzotriazole (HoBt; manufactured by Dojindo; N Wako Pure Chemical Industries) were prepared. Among these, folic acid and dopamine hydrochloride were mixed so that the original concentration was 1/4, and the others were 1/10 of the original concentration, and the solution was adjusted to 20 ml with DMF. The mixed solution was reacted at room temperature for 20 hours while gently stirring. A part of the reaction solution was diluted 10-fold with ultrapure water, and this solution was subjected to reverse phase chromatography (HPLC system: Prominence (manufactured by Shimadzu Corporation), column: Chromolith RP-18e 100-3 mm (manufactured by Merck), mobile phase: A Analysis was performed using methanol (Wako Pure Chemical Industries) B 0.1% trifluoroacetic acid aqueous solution (Wako Pure Chemical Industries, flow rate: 2 ml / min). As a result of setting the wavelength to 210 nm with an ultraviolet detector and performing gradient elution so that methanol becomes 100% in 1 to 10 minutes after injection (0.02 ml), a peak considered to be a complex of folic acid and dopamine hydrochloride in the vicinity of 9 minutes It was confirmed. Moreover, the peak of each of folic acid and dopamine hydrochloride was below the detection limit. From these, the formation of a complex of folic acid and dopamine hydrochloride was confirmed. Next, the remainder of the reaction solution was concentrated 10 times under reduced pressure to obtain a reaction concentrated solution. The titanium oxide composite particles obtained in Example 1 were adjusted to 1% solid components with ultrapure water, and 1/10 amount of the reaction concentrated solution was mixed therewith to make 1 ml in total. The mixed solution was reacted at room temperature for 1 hour while gently stirring. After the reaction, the precipitated component was centrifuged (1500 g, 10 min) to collect the supernatant, and 1 ml of this solution was subjected to water 1. using a natural drop column NAP-10 (manufactured by GE Healthcare Bioscience) for buffer exchange. Collected in 5 ml, the unreacted folic acid-dopamine hydrochloride complex and DMF were removed. Removal of the folic acid-dopamine hydrochloride complex and DMF was confirmed by reverse phase chromatography in the same manner as described above, and it was confirmed that there was no free folic acid-dopamine hydrochloride complex. From these, the production of folic acid and dopamine hydrochloride complex-bound titanium oxide functional molecular complex particles was confirmed.

Example 18: Confirmation of UV irradiation and fluorescence on complex-bound titanium oxide functional molecular composite particles of folic acid and dopamine hydrochloride The composite-bound titanium oxide functional molecular composite particles of folic acid and dopamine hydrochloride obtained in Example 17 The solution was prepared in 3 ml using phosphate buffered saline so that the titanium oxide concentrations were 0.05, 0.01, and 0.005%, respectively. While stirring these solutions, irradiation was performed at an ultraviolet intensity of 0.9 mW / cm ² using an ultraviolet lamp with a wavelength of 352 nm (FL20S · BLB-A; manufactured by Toshiba Lighting & Technology Corp.), and at 0, 3, 6, and 9 min after the start of irradiation. The fluorescence intensity of the solution was set to an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm using a fluorescence spectrophotometer (RF-5300PC; manufactured by Shimadzu Corporation), and measurement was performed. The results are shown in FIG. It was confirmed that the fluorescence intensity increased with increasing irradiation time and titanium oxide concentration. This was considered to be fluorescence derived from the pterin compound liberated from folic acid with the reaction with radicals generated by ultraviolet irradiation of this solution.

Example 19: Binding of methionine to dihydroxyphenylpropionic acid-bonded titanium oxide functional molecule composite particles The solution of dihydroxyphenylpropionic acid-bonded titanium oxide functional molecular composite particles obtained in Example 13 and 1-ethyl-3- (3-Dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Dojindo) was adjusted to be mixed using a 50 mM borate buffer solution (pH 9.0) so that the concentrations were 15 mg / ml and 80 mM, respectively. The prepared solution was mixed with methionine (manufactured by Wako Pure Chemical Industries) to a concentration of 10 mM to obtain 0.2 ml of a solution. This solution was allowed to react with shaking at room temperature for 24 hours. 50 μl of the reacted solution was adjusted to 1000-fold dilution with ultrapure water, and this was added 1/10 to 50 mM borate buffer to make 440 μl. Further, 20 μl of 0.3 mg / ml fluorescamine (manufactured by Tokyo Chemical Industry Co., Ltd.) dissolved in acetone was added and the solution was stirred. Then, the fluorescence value was measured with a fluorometer (manufactured by Shimadzu Corporation: RF-5300) as follows. Measured under conditions. (Wavelength: Ex = 395 nm / Em = 495 nm, Bandwidth: Ex = 5 nm / Em = 10 nm, Sensitivity: High, Response: Auto). Unbound methionine was calculated using a calibration curve prepared in advance, and the rate of concentration change from the initial concentration, that is, the rate of binding was calculated. As a result, it was 40.2%. From the above, it was confirmed that methionine was bound via dihydroxyphenylpropionic acid in dihydroxyphenylpropionic acid-bonded titanium oxide functional molecule composite particles.

Example 20: Binding of radical-responsive molecule to dopamine-bonded titanium oxide functional molecule composite particle The dopamine-bonded titanium oxide functional molecule composite particle obtained in Example 12 was 0.3% in final concentration, and 2, 3 -Dihydro-6-isothiocyanato-1,4-phthalazedione (manufactured by Dojindo) was mixed in 20 mM phosphate buffer (pH 7.0) to a final concentration of 1 mM and adjusted to 3 ml. This solution was allowed to stand for 24 hours at 4 ° C. in the dark and subjected to a binding reaction. Unreacted substances were removed by recovering 2.5 ml of this solution with 3.5 ml of water using a PD-10 (GE Healthcare Bioscience) natural drop column for buffer exchange. From these facts, the radical-responsive molecule-bonded titanium oxide functional molecule composite particle in which 2,3-dihydro-6-isothiocyanato-1,4-phthalazedione, which is a radical-responsive molecule, is bonded to the titanium oxide composite particle. The production was confirmed.

Example 21: Binding of radical-responsive molecule to dopamine-bound titanium oxide functional molecule composite particle 2
The dopamine-bonded titanium oxide functional molecular complex particles obtained in Example 12 were 0.3% in final concentration, and 2 ′, 7′-dichlorohydrofluorescein diacetate, succinimidyl ester (manufactured by Invitrogen) was adjusted to 1 mM at final concentration. Mix in 20 mM phosphate buffer (pH 7.0) to adjust to 3 ml. This solution was allowed to stand for 24 hours at 4 ° C. in the dark and subjected to a binding reaction. Unreacted substances were removed by recovering 2.5 ml of this solution with 3.5 ml of water using a PD-10 (GE Healthcare Bioscience) natural drop column for buffer exchange. From these facts, production of fluorescent molecule-bound titanium oxide functional molecule composite particles in which 2 ', 7'-dichlorodihydrofluorescein diacetate and succinimidyl ester, which are radical-responsive molecules, were bound to the titanium oxide composite particles was confirmed.

Example 22: Binding of ferrocenecarboxylic acid and dopamine to titanium oxide composite particles Dimethylformamide (DMF; Wako Pure Chemical Industries) so that ferrocenecarboxylic acid (manufactured by Wako Pure Chemical Industries) and dopamine hydrochloride (manufactured by Wako Pure Chemical Industries) become 1 mM. (Made by industry). Similarly, using DMF, 200 mM Benzotriazole-1-yl-oxy-trispyrrolophosphonophosphate phosphophosphate (PyBop; manufactured by Merck), 200 mM 1-hydroxybenzotriazole (HoBt; manufactured by Dojindo; N Wako Pure Chemical Industries) were prepared. Of these, ferrocenecarboxylic acid and dopamine hydrochloride were mixed so as to be 1/4 of the original concentration, and the others were 1/10 of the original concentration, and the solution was adjusted to 20 ml with DMF. The mixed solution was reacted at room temperature for 20 hours while gently stirring. A part of the reaction solution was diluted 10-fold with ultrapure water, and this solution was subjected to reverse phase chromatography (HPLC system: Prominence (manufactured by Shimadzu Corporation), column: Chromolith RP-18e 100-3 mm (manufactured by Merck), mobile phase: A Analysis was performed using methanol (Wako Pure Chemical Industries) B 0.1% trifluoroacetic acid aqueous solution (Wako Pure Chemical Industries, flow rate: 2 ml / min). As a result of setting the wavelength to 210 nm with an ultraviolet ray detector and performing gradient elution so that methanol becomes 100% in 1 to 10 minutes after injection (0.02 ml), a peak considered to be a complex of ferrocenecarboxylic acid and dopamine hydrochloride was obtained. confirmed. In addition, the single peaks of ferrocenecarboxylic acid and dopamine hydrochloride were below the detection limit. From these facts, formation of a complex of ferrocenecarboxylic acid and dopamine hydrochloride was confirmed.

  The remainder of the reaction solution was concentrated 10 times under reduced pressure to obtain a reaction concentrated solution. The titanium oxide composite particles obtained in Example 1 were adjusted to 1% solid components with ultrapure water, and 1/10 amount of the reaction concentrated solution was mixed therewith to make 1 ml in total. The mixed solution was reacted at room temperature for 1 hour while gently stirring. After the reaction, the precipitated component was centrifuged (1500 g, 10 min) to collect the supernatant, and 1 ml of this solution was subjected to water 1. using a natural drop column NAP-10 (manufactured by GE Healthcare Bioscience) for buffer exchange. Collected in 5 ml, the unreacted ferrocenecarboxylic acid-dopamine hydrochloride complex and DMF were removed. The absorption spectrum of the wavelength in the visible light region (400 nm) of this solution was confirmed by an ultraviolet-visible light spectrophotometer (UV1600; manufactured by Shimadzu Corporation), and an increase was observed, so that a complex of ferrocenecarboxylic acid and dopamine hydrochloride was bound. It was thought that it was. From these, the production of composite-bound titanium oxide functional molecular composite particles of ferrocenecarboxylic acid and dopamine hydrochloride was confirmed.

Example 23: Binding of fluorescent molecules to dopamine-bound titanium oxide functional molecule composite particles 2
The dopamine-bonded titanium oxide functional molecule complex particles obtained in Example 12 were 20% phosphoric acid so that the final concentration was 0.3%, and Cy7-NHS ester (manufactured by GE Healthcare Bioscience) was 1 mM at the final concentration. Mix in buffer (pH 7.0) to adjust to 3 ml. This solution was allowed to stand for 24 hours at 4 ° C. in the dark and subjected to a binding reaction. Unreacted substances were removed by recovering 2.5 ml of this solution with 3.5 ml of water using a PD-10 (GE Healthcare Bioscience) natural drop column for buffer exchange. As a result of measuring the fluorescence intensity of this solution with a fluorescence spectrophotometer (RF-5300PC; manufactured by Shimadzu Corporation) at an excitation wavelength of 743 nm and a fluorescence wavelength of 767 nm, it was confirmed that fluorescence was exhibited. From these facts, production of fluorescent molecule-bound titanium oxide functional molecule composite particles in which Cy7-NHS ester, which is a fluorescent molecule, was bound to titanium oxide composite particles was confirmed.

Example 24: Catechol binding 2 to titanium oxide composite particles 2
Each titanium oxide composite particle obtained in Examples 8 to 10 was made 1% solid component with ultrapure water. Next, catechol was adjusted with ultrapure water to a molar concentration of 100 mM, 10 mM, 1 mM, and 0.1 mM. Equal amounts of each catechol solution are mixed with 1% solution of titanium oxide composite particles, and finally the titanium oxide composite particles and the catechol solution are finished with phosphate buffered saline (PBS; pH 7.4). The concentration was adjusted to 10-fold dilution. Each of the adjusted solutions was transferred to a thermostat and set to 40 ° C., and a binding reaction was performed for 3 hours. When the absorption spectrum of the wavelength in the visible light region of the solution after the reaction was confirmed by an ultraviolet-visible light spectrophotometer, an increase was observed for each solution, and thus it was considered that catechol was bound.

It is a figure which shows an example of the titanium oxide functional molecule composite particle | grains of this invention. It is a figure which shows the relationship between the ultraviolet irradiation time of the titanium oxide functional molecule composite particle obtained in Example 17, and the fluorescence intensity measured in Example 18.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Titanium oxide particle 2 ... Water-soluble polymer 3 ... Functional molecule

Claims (18)

Titanium oxide particles,
It comprises a water-soluble polymer that is bonded to the surface of the titanium oxide particles via at least one functional group selected from the group consisting of a carboxyl group, an amino group, a diol group, a salicylic acid group, and a phosphoric acid group. , Titanium oxide composite particles,
A functional molecule formed by further bonding to the surface of the titanium oxide composite particles;
The functional molecule is
(1) having at least one functional group selected from the group of carboxyl group, amino group, diol group, salicylic acid group, and phosphoric acid group,
(2) a) a saturated or unsaturated chain hydrocarbon group having 6 to 40 carbon atoms, b) a saturated or unsaturated 5- to 6-membered heterocyclic group having or not having a substituent, or c) A compound comprising a saturated or unsaturated 5- or 6-membered cyclic hydrocarbon group with or without a substituent,
Titanium oxide functional molecular composite particles, which are bonded to the titanium oxide via the functional group. The binding amount of the functional molecule is 1.0 × 10 ^{−1 to} 1.0 × 10 ⁻⁵ parts by mass / part by mass of titanium oxide particles per mass of the titanium oxide particles. 2. Titanium oxide functional molecular composite particles according to 1.   The titanium oxide functional molecule composite particle according to claim 1, wherein the functional molecule is a catechol.   4. The titanium oxide functional molecule composite particle according to claim 3, wherein the functional molecule is at least one selected from the group consisting of catechol, dopamine, and dihydroxyphenylpropionic acid.   The titanium oxide functional molecule composite particle according to claim 1, wherein the functional molecule is a radioactive substance. The titanium oxide functional molecule composite particle according to claim 5, wherein the radioactive substance is ¹⁴ C-labeled catechol.   The titanium oxide functional molecule composite particle according to claim 1, wherein the functional molecule comprises a radical-responsive molecule.   The titanium oxide functional molecule composite particle according to claim 1, wherein the functional molecule comprises a fluorescent molecule.   The titanium oxide functional molecule composite particle according to claim 1 or 2, wherein the functional molecule comprises aminolevulinic acid.   The titanium oxide functional molecule composite particle according to claim 1, wherein the functional molecule comprises ferrocene.   The titanium oxide functional molecular composite particle according to any one of claims 1 to 10, wherein the water-soluble polymer has a weight average molecular weight of 5000 to 40000.   The titanium oxide function according to claim 1, wherein the water-soluble polymer comprises at least one selected from the group consisting of polyethylene glycol, polyacrylic acid, and polyethyleneimine. Sex molecule composite particles.   The titanium oxide functional molecule composite particle according to any one of claims 1 to 12, which has a particle diameter of 20 to 200 nm.   A dispersion comprising the titanium oxide functional molecule composite particle according to any one of claims 1 to 13 and a solvent in which the particle is dispersed.   The dispersion according to claim 14, wherein the solvent is an aqueous solvent.   The dispersion according to claim 14 or 15, wherein the pH of the solvent is 5 to 8.   The dispersion according to any one of claims 14 to 16, wherein the solvent is physiological saline.   The dispersion according to any one of claims 14 to 17, wherein the titanium oxide functional molecule composite particles are contained in an amount of 0.001 to 1 mass%.

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