JP5224531B2 - Treatment for bladder cancer - Google Patents

Description

  The present invention relates to a titanium oxide complex dispersed in an aqueous solvent with a water-soluble polymer, by binding a linker molecule to the titanium oxide surface without altering the water-soluble polymer, and further via the linker molecule. The present invention relates to a therapeutic agent for bladder cancer, which is a titanium oxide-antibody complex having an antibody bound thereto and having catalytic activity by irradiation with ultrasonic waves.

  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 an anionic polymer, thereby exhibiting a stable dispersibility even in neutral physiological saline close to the in vivo environment, and having a photocatalytic activity function during ultraviolet irradiation It is.

  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.

  Bladder cancer is a common cancer in the urological area. It is known that the degree of invasion (also called muscle layer invasion) correlates with the frequency of lymph nodes and distant metastases in bladder cancer. 70% to 80% of bladder cancer is superficial cancer without invasion. Patients with superficial bladder cancer undergo transurethral tumor resection, and some patients are subsequently treated with intravesical immunotherapy / chemotherapy. As a result, the 5-year survival rate for patients with superficial bladder cancer is close to 90%. However, it is known that 50% to 70% of patients with superficial bladder cancer will relapse, and 5-20% of them will progress to bladder cancer infiltrating the muscle layer. The cause of recurrence is that bladder cancer is often multiple, and bladder cancer cells that cannot be removed by treatment such as surgery remain. For the purpose of preventing recurrence, treatment with immunotherapy / chemotherapy may be performed. For example, in BCG intravesical infusion therapy, since it also acts on a normal site, it may have side effects such as cystitis. Is almost. Once a patient has relapsed, the recurrence may be repeated, and eventually the entire bladder may not be removed. Therefore, improvement of the patient's QOL (quality of life) in the treatment of bladder cancer is required.

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

  The present invention relates to a titanium oxide complex that retains dispersibility in an aqueous solvent by a water-soluble polymer and has catalytic activity by irradiation with ultrasonic waves, through a linker molecule without altering the water-soluble polymer. The purpose of the present invention is to provide a therapeutic agent for bladder cancer, which is a titanium oxide-antibody complex imparted with a selective binding ability without losing dispersibility and catalytic activity.

  The present inventors have recently made it possible to disperse the water-soluble polymer without altering the water-soluble polymer by bonding a linker molecule to the titanium oxide surface of the titanium oxide composite dispersed in an aqueous solvent. It was found that it is possible to newly apply an antibody while maintaining catalytic activity.

  That is, according to the bladder cancer therapeutic agent of the present invention, a linker molecule is bound to the titanium oxide surface of a titanium oxide complex dispersed in an aqueous solvent with a water-soluble polymer, and further an antibody is bound via the linker molecule. It is a titanium oxide-antibody complex particle that retains high dispersibility without altering the water-soluble polymer, has catalytic activity due to ultrasonic irradiation, and can bind to an antigen. A therapeutic agent for bladder cancer can be prepared. By using an antibody that specifically binds to the surface antigen of the bladder cancer cell as the antibody, and by irradiating the bladder cancer therapeutic agent with the surface antigen of the bladder cancer cell that is the antigen, ultrasonic irradiation, It can be expected that the generated radical species act on bladder cancer cells and enhance the therapeutic effect. Furthermore, by performing weak ultrasonic irradiation that does not adversely affect the surrounding normal cells, the effect is exerted only in the local area where the bladder cancer therapeutic agent is accumulated, and side effects can be expected to be reduced. In addition, this ultrasonic irradiation can treat multiple bladder cancers by irradiating a wide area of the bladder surface, and also acts on bladder cancer cells that cannot be removed by treatment such as surgery. The effect of preventing recurrence can be expected. Therefore, the bladder cancer therapeutic agent of the present invention can be used as an agent for promoting ultrasonic bladder cancer treatment performed by accumulating in an affected area and further irradiating ultrasonic waves.

The bladder cancer therapeutic agent of the present invention is bound to the surface of the titanium oxide particles via at least one functional group selected from the group of carboxyl group, amino group, diol group, salicylic acid group, and phosphoric acid group. A titanium oxide composite comprising a water-soluble polymer,
A linker molecule bonded to the surface of the titanium oxide complex;
A therapeutic agent for bladder cancer, which is a titanium oxide-antibody complex comprising an antibody bound via the linker molecule and having catalytic activity by ultrasonic irradiation,
The bladder cancer therapeutic agent is administered by being injected into the bladder and bound to the surface antigen of the bladder cancer cell, which is an antigen, and the unbound bladder cancer therapeutic agent is removed by urination or intravesical washing. It is a feature.

  The dispersion according to the present invention comprises a therapeutic agent for bladder cancer and a solvent in which the therapeutic agent for bladder cancer is dispersed.

  According to the present invention, a titanium oxide composite that maintains dispersibility in an aqueous solvent by a water-soluble polymer and has catalytic activity due to ultrasonic irradiation can be mediated through a linker molecule without altering the water-soluble polymer. Thus, it is possible to provide a therapeutic agent for bladder cancer, and a dispersion thereof, which is a titanium oxide-antibody complex imparted with a selective binding ability without losing dispersibility and catalytic activity by modifying the antibody.

  The bladder cancer therapeutic agent according to the present invention comprises a bladder cancer therapeutic agent or a dispersion in which a bladder cancer therapeutic agent is dispersed.

It is a figure which shows an example of the bladder cancer therapeutic agent of this invention. It is a figure which shows the fluorescence intensity through the fluorescence reagent for a singlet oxygen detection resulting from generation | occurrence | production of the singlet oxygen by ultrasonic irradiation measured about various particles in Example 9. In Example 10, it is a figure which shows the sensorgram obtained by measuring with a surface plasmon resonance method about a titanium oxide antibody complex. It is a figure which shows the fluorescence intensity through the fluorescent reagent for hydroxy radical detection resulting from the generation | occurrence | production of the hydroxy radical after ultrasonic irradiation measured about the titanium oxide composite E in Example 15. In Example 18, it is a figure which shows the fluorescence intensity distribution with respect to the cell number accompanying the coupling | bonding to the tumor cell measured by the flow cytometry method about the test solution A, the test solution B, and control.

  The therapeutic agent for bladder cancer of the present invention includes a titanium oxide-antibody complex comprising titanium oxide particles, a water-soluble polymer, a linker molecule, and an antibody. FIG. 1 shows an example of a bladder cancer therapeutic agent. As shown in FIG. 1, the composite particle is a particle in which a water-soluble polymer 2 and a linker molecule 3 are bonded to the surface of a titanium oxide particle 1 and an antibody 4 is bonded through the linker molecule 3. The bond between the titanium oxide particles 1 and the water-soluble polymer 2 is formed through 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.

  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. It is also possible to retain antibody binding via a linker molecule. 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 ultrasonic irradiation, 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 charge or hydration, and the bladder cancer of the present invention The therapeutic agent can be dispersed. A method for introducing an antibody to a water-soluble polymer bound to the surface of titanium oxide particles is known. In such a case, in order to chemically bond the water-soluble polymer and the antibody, 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 antibody 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 particles changes before and after the antibody binding. 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 antibody bound through the linker molecule bound to the surface of the titanium oxide particles in the present invention can retain high dispersibility due to the water-soluble polymer by binding without altering the water-soluble polymer. For this reason, it is possible to design a molecule with a high degree of freedom when binding an antibody without considering dispersibility change caused by alteration of the water-soluble polymer.

  According to the bladder cancer therapeutic agent of the present invention, a linker molecule is bound to the titanium oxide surface of a titanium oxide complex dispersed in an aqueous solvent with a water-soluble polymer, and an antibody is further bound via the linker molecule. Thus, a bladder cancer therapeutic agent characterized by being a titanium oxide-antibody complex that retains high dispersibility without altering the water-soluble polymer can be produced. By binding the antibody in this way, the therapeutic agent for bladder cancer of the present invention can bind to the antigen. In addition, radical species can be generated by irradiating the therapeutic agent for bladder cancer of the present invention with ultrasonic waves. In general, radical species are highly reactive but have a short lifetime, and they diffuse slightly and react with nearby substances. For this reason, it can be expected that the reactivity between the radical species and the antigen is enhanced by irradiating with ultrasound in the state where the therapeutic agent for bladder cancer of the present invention is bound to the antigen. By using an antibody that specifically binds to the surface antigen of the bladder cancer cell as the antibody, and by irradiating the bladder cancer therapeutic agent with the surface antigen of the bladder cancer cell that is the antigen, ultrasonic irradiation, It can be expected that the generated radical species act on bladder cancer cells and enhance the therapeutic effect. Furthermore, by performing weak ultrasonic irradiation that does not adversely affect the surrounding normal cells, the effect is exerted only in the local area where the bladder cancer therapeutic agent is accumulated, and side effects can be expected to be reduced. In addition, this ultrasonic irradiation can treat multiple bladder cancers by irradiating a wide area of the bladder surface, and also acts on bladder cancer cells that cannot be removed by treatment such as surgery. The effect of preventing recurrence can be expected. Therefore, the bladder cancer therapeutic agent of the present invention can be used as an agent for promoting ultrasonic bladder cancer treatment performed by accumulating in an affected area and further irradiating ultrasonic waves.

  Further, according to the bladder cancer therapeutic agent of the present invention, a water-soluble polymer can be obtained by binding a photoresponsive molecule or radioactive substance via a linker molecule to the titanium oxide surface of the titanium oxide-antibody complex. High dispersibility can be maintained without alteration. In particular, radioactive materials need to be used in as few steps as possible from the viewpoint of safety, but radioactive materials on the surface of titanium oxide are compared to titanium oxide composites dispersed in water-based solvents with water-soluble polymers. After binding, the particles can be labeled in a simple and few steps by simply separating and removing unbound radioactive material 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 therapeutic agent for bladder cancer of the present invention is also used as a tracer experimental material for confirming pharmacokinetics after administration into the body, or as a medical material for diagnosis and treatment performed by irradiating the affected area with ultrasonic waves. Is possible.

  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 deactivation that may occur when the surface is covered with the polymer can be suppressed, and the catalytic activity of the titanium oxide particles can be sufficiently exhibited.

  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-antibody complex can be dispersed in an aqueous solvent. Among the water-soluble polymers used in the present invention, those having a charge are anionic or cationic water-soluble polymers, and those having no charge and imparting dispersibility by hydration are nonionic. A water-soluble polymer having at least one of them.

  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-antibody complex is in a neutral aqueous solvent in which it is difficult to disperse the titanium oxide particles due to the charge or hydration action of the water-soluble polymer. Can be 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 water-soluble polymer having anionic property can disperse the bladder cancer therapeutic agent of the present invention in an aqueous solvent. It is. As the water-soluble polymer having an anionic property, those having a plurality of carboxyl groups can be suitably used. For example, carboxymethyl starch, carboxymethyl dextran, carboxymethyl cellulose, polycarboxylic acids, and a copolymer having a carboxyl group unit. (Copolymer) and the like. 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 water-soluble polymer having a cationic property can disperse the bladder cancer therapeutic agent of the present invention in an aqueous solvent. It is. The water-soluble polymer having a cationic property can be suitably used as a polymer having a plurality of amino groups, and examples thereof include polyamino acids, polypeptides, polyamines, and copolymers (copolymers) having amine units. It is done. 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, the water-soluble polymer used in the present invention can be any nonionic water-soluble polymer as long as the bladder cancer therapeutic agent of the present invention can be dispersed in an aqueous solvent. It is. The water-soluble polymer having nonionic properties is preferably a polymer having a hydroxyl group and / or a polyoxyalkylene group. 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.

  The water-soluble polymers exemplified above can be freely combined with each of the elements described so far as long as the dispersibility of the therapeutic agent for bladder cancer of the present invention is not hindered.

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

  According to a preferred embodiment of the present invention, the linker 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 5- or 6-membered heterocyclic group, or c) a saturated or unsaturated 5- or 6-membered cyclic hydrocarbon group having or not having a substituent.

  The linker molecule having the above carbon number has a smaller molecular size than the water-soluble polymer. The linker molecule is bonded to the titanium oxide surface. For this reason, in the titanium oxide-antibody complex of the present invention, the water-soluble polymer is located in the outer shell, whereas it has a structure having a linker molecule at a more internal position. The outer shell has the greatest influence on the dispersibility of the therapeutic agent for bladder cancer of the present invention. That is, the water-soluble polymer located in the outer shell has less influence on the dispersibility of the linker molecule located inside, and can be suitably used.

The amount of the linker molecule bound to the bladder cancer therapeutic agent of the present invention is 1.0 × 10 ^{−6 to} 1.0 × 10 ⁻³ mol per gram of titanium oxide particles, more preferably 1.0. × 10 ^{−6 to} 1.0 × 10 ⁻⁴ mol / titanium oxide particles-g. Within this range, the therapeutic agent for bladder cancer of the present invention can be preferably used because it can be dispersed in a 10% protein solution that is close to the in vivo environment as a solvent. Furthermore, when it is within this range, the therapeutic agent for bladder cancer of the present invention can be suitably used because it has catalytic activity and can generate radical species when irradiated with ultrasonic waves.

  Examples of such linker 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, Examples thereof include catechols 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, as a molecule | numerator which has an alkyl structure, the molecule | numerator which has alkyl groups, such as a hexyl group, an octyl group, a lauryl group, a palmityl group, a stearyl group, is mentioned. Alternatively, an alkenyl group such as a hexenyl group, an octenyl group, or an oleyl group, or a saturated or unsaturated aliphatic hydrocarbon group such as a cycloalkyl group can be used.

  The linker molecule and the amount thereof exemplified above can be suitably combined with each component described so far.

  As used herein, "antibody" is intended to be an immunoglobulin (IgA, IgD, IgE, IgG, IgM and their Fab fragments, F (ab ') 2 fragments, Fc fragments), as an example Include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies and anti-idiotype antibodies. Monoclonal antibodies are preferred from the standpoint of specific binding ability and quality control in drug production. Moreover, these production methods are not particularly limited.

  According to a preferred embodiment of the present invention, the antibody that binds via the linker molecule is preferably an antibody that specifically binds to the surface antigen of bladder cancer cells. More specifically, anti-EpCAM antibody, anti-CD44 antibody, anti-EGF receptor antibody and the like can be mentioned.

  In addition, in order to positively accumulate the bladder cancer therapeutic agent of the present invention at the bladder cancer site, the molecule that binds via the linker molecule is not limited to the antibody, but specifically binds to, for example, the surface antigen of bladder cancer cells. It may be a peptide or amino acid sequence. 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. There is no restriction | limiting in particular as a nucleic acid, Nucleobases, such as DNA and RNA, Peptide nucleic acids, such as PNA, or an aptamer etc. in which they form a higher-order structure can also be used. The peptides and amino acid sequences described above may be used in combination with antibodies. Moreover, these peptides and amino acid sequences can be suitably combined with each component described so far.

  According to a preferred embodiment of the present invention, in addition to an antibody that binds via a linker molecule, another functional molecule may be bound via a linker molecule. An example of the functional molecule is a photoresponsive molecule, and a fluorescent molecule can be used as the photoresponsive molecule.

Moreover, a radioactive compound is mentioned as another example of a functional molecule. The radioactive compounds include compounds containing an isotope element, for example, ^{14 C-labeled} catechol with ^{14 C-is} preferably used.

  Another example of a functional molecule is a radical responsive compound. The radical-responsive compound includes a chemiluminescent molecule, a fluorescent molecule, or a spin trap agent that exhibits specific reactivity with a radical. 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, carboplatin, etoposide, nedaplatin, paclitaxel, At least one anticancer agent from docetaxel, irinotecan hydrochloride, etc., antibacterial agents such as penicillin, macrolide, new quinolone, tetracycline, lamivudine, nelfinavir, indinavir, saquinavir, interferon, amantadine, acyclovir and other antiviral agents, And hormonal disease treatments such as neuprorelin, buserelin, goserelin, triptorelin, nafarelin, ibuprof Analgesics such as emissions and the like.

  Another example of a functional molecule is a molecule containing a low-valent transition metal. Low-valent transition metals are known to generate hydrogen radicals by decomposing hydrogen peroxide by the Harber-Weiss mechanism (chemicals of reactive oxygen species [quarterly published Chemical Review No. 7] edited by The Chemical Society of Japan) For example, when a divalent iron ion is used as the valence transition metal, it is well known as the Fenton reaction. In addition, various radicals including hydroxy radicals have a cytotoxic effect. Therefore, if molecules containing these low-valent transition metals are bonded via a linker molecule, radicals can be generated as long as hydrogen peroxide is present, and the cytotoxic action can be sustained. That is, even after the ultrasonic irradiation is stopped, the hydrogen peroxide accumulated in the system and the oxidizing power is stronger due to the Fenton reaction between the molecule containing the low-valent transition metal bonded to the bladder cancer therapeutic agent of the present invention. It is possible to continuously generate hydroxy radicals and obtain a continuous antitumor effect associated therewith. However, when a complex is used as a molecule containing a low-valent transition metal, not only a free hydroxy radical but also a ferryl complex that can be generated when an iron complex is used, for example, a so-called Crypto-HO. Involvement is also possible. Examples of such low-valent transition metals include trivalent titanium, divalent chromium, and monovalent copper in addition to divalent iron. Furthermore, examples of the molecule containing such a low-valent transition metal include ferrocenecarboxylic acid, a complex of bicinchoninic acid and monovalent copper, and the like.

  The functional molecules described above can be suitably combined with the constituent elements described so far to achieve the various effects described above without impeding the effects of the present invention.

  According to a preferred embodiment of the present invention, the linker molecule used in the present invention is a molecule in which the functional molecule and the functional group that binds to the titanium oxide surface are further bonded via another linker. Absent.

  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] propionyl hydrazide, 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] Succinimide ester, chlorotriazine, dichlorotriazine, trichlorotriazine, succinimidyl-4-hydrazinonicotinate-acetone hydrazone, C6-succinimidyl-4-hydrazinonicotinate-acetone hydrazone, succin Mijiru -4 hydrazide terephthalate - hydrochloride, succinimidyl-4- formyl benzoate, C6- succinimidyl-4- formylphenyl benzoate and the like. Further, the linker may be composed of a plurality of types of linkers such that other linkers are bonded to each other. The above linker can be suitably combined with each component described so far.

  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 linker molecule is preferably an enediol group, more preferably an α-diol group. . 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 high 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. The use of anatase-type titanium oxide or rutile-type titanium oxide as the titanium oxide particles can be suitably combined with the constituent elements described so far, and the above-described new effects can be achieved.

  According to a preferred embodiment of the present invention, the therapeutic agent for bladder cancer of the present invention has a particle size of 20 to 200 nm, more preferably 50 to 200 nm, still more preferably 50 to 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 an ultrasonic wave of 400 kHz-20 MHz. Therefore, cancer tissue can be killed with high efficiency by ultrasonic irradiation.

  According to another preferred embodiment of the present invention, when the bladder cancer therapeutic agent has 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.

  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).

  Making the therapeutic agent for bladder cancer of the present invention to have the above particle diameter can be suitably combined with each of the constituent elements described so far, and the above-described new effect can be achieved.

  The therapeutic agent for bladder cancer of the present invention includes not only a single type of titanium oxide composite but also a mixture or composite of multiple 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 bladder cancer therapeutic agent of the present invention is preferably dispersed in a solvent to be in the form of a dispersion. Thereby, the therapeutic agent for bladder cancer of the present invention can be used as a drug that is efficiently administered into a patient's body by various methods such as infusion, 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 therapeutic agent for bladder cancer of the present invention is preferably contained in an amount of 0.001-1% by mass or less, more preferably 0.001-0.1% by mass, based on 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 therapeutic agent for bladder cancer of the present invention can be administered into a patient's body by various methods such as infusion, injection, and application. In particular, when used by intravenous or subcutaneous administration routes, the so-called DDS is utilized by utilizing the EPR effect due to the size of the particles, the retention in the blood, and the interaction between the antibody bound to the particles and the antigen derived from the affected area. It is preferable from the viewpoint of reducing the burden on the patient by an effective treatment. The therapeutic agent for bladder cancer administered into the body reaches the cancer tissue and accumulates like a drug delivery system.

  When the therapeutic agent for bladder cancer of the present invention is used in a route of administration via a blood vessel or organ close to the affected area, the dispersibility between the in vivo environment and the antibody bound to the particles and the antigen derived from the affected area The action is preferable from the viewpoint of reducing the burden on the patient by so-called local DDS treatment. The therapeutic agent for bladder cancer administered into the body reaches the cancer tissue and accumulates like a drug delivery system.

  In order to treat bladder cancer using the bladder cancer therapeutic agent of the present invention, the bladder cancer therapeutic agent of the present invention is injected into the urinary bladder, and the bladder cancer therapeutic agent that has not been bound by urination for a certain period of time or intravesical lavage is removed. After that, the bladder cancer therapeutic agent is irradiated with a weak ultrasonic wave that does not adversely affect the surrounding normal cells in a state in which the bladder cancer therapeutic agent is bound to the surface antigen of the bladder cancer cell as an antigen. It is effective only in the local area where is accumulated, and can be expected to reduce side effects. In addition, this ultrasonic irradiation can treat multiple bladder cancers by irradiating a wide area of the bladder surface, and also acts on bladder cancer cells that cannot be removed by treatment such as surgery. The effect of preventing recurrence can be expected.

  The therapeutic agent for bladder cancer of the present invention is irradiated with ultrasonic waves or ultraviolet rays, preferably ultrasonic waves, and can be rendered cytotoxic by the irradiation. This therapeutic agent for bladder cancer can be killed by being administered into the body, receiving ultrasound irradiation, and generating cytotoxic cells by the irradiation, but not only in the body but also in a test tube. A cell can be killed. In the present invention, the subject to be killed is preferably bladder cancer cells. That is, the bladder cancer therapeutic agent of the present invention can be used as a drug that is activated by irradiation with ultrasonic waves or ultraviolet rays to kill bladder cancer cells.

  According to a preferred embodiment of the present invention, ultrasonic treatment is performed on a cancer tissue in which the bladder cancer therapeutic agent of the present invention is 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 it is used in combination with the bladder cancer therapeutic agent of the present invention to treat an affected part or target site that exists in the deep part of the living body in a non-invasive state. Can be realized. Furthermore, the bladder cancer therapeutic agent of the present invention accumulates on the affected part or target site, so that only the local area where the bladder cancer therapeutic agent of the present invention is accumulated with weak ultrasonic waves that do not adversely affect the surrounding normal cells. Can act.

  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, the biological killing effect given by these semiconductor particles is in the qualitative and quantitative increase of radical species, and it is considered that these radical species become cytotoxins. 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.

Production method The titanium oxide complex used in the therapeutic agent for bladder cancer of the present invention is a water-soluble polymer having 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. Can be produced by bonding to the titanium oxide particles. The production of the titanium oxide composite by this method is carried out, for example, in an aprotic solvent with at least one functional group selected from titanium oxide particles and a carboxyl group, amino group, diol group, salicylic acid group, and phosphoric acid group. The nonionic water-soluble polymer having a group is dispersed, and the obtained dispersion is heated at 80 to 220 ° C., for example, for 1 to 16 hours. Examples of preferred aprotic solvents include dimethylformamide, dioxane, and dimethyl sulfoxide.

  The therapeutic agent for bladder cancer of the present invention is obtained from the group of carboxyl group, amino group, diol group, salicylic acid group, and phosphoric acid group on the titanium oxide surface of the titanium oxide complex dispersed in an aqueous solvent with a water-soluble polymer. It can be produced by forming a titanium oxide-antibody complex in which a linker molecule is bound via at least one selected functional group and the antibody is further modified via the linker molecule. Production of a bladder cancer therapeutic agent by this method includes, for example, a titanium oxide complex and at least one functional group selected from a carboxyl group, an amino group, a diol group, a salicylic acid group, and a phosphate group in an aqueous solution. For example, by heating at 0 ° C. to 50 ° C. for 1 to 16 hours, removing the unbound linker molecule by membrane separation or the like, and then the amino group or the like of the linker molecule bound to the titanium oxide complex After reacting the functional group with a carbodiimide agent such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and activating the functional group, the unreacted carbodiimide agent is removed by membrane separation or the like, and the antibody is mixed. For example, the reaction can be performed at 0 ° C. to room temperature for 1 to 16 hours, and unreacted antibody is removed by membrane separation or the like.

  Alternatively, the therapeutic agent for bladder cancer of the present invention may be a linker molecule via an antibody and at least one functional group selected from the group of carboxyl group, amino group, diol group, salicylic acid group, and phosphoric acid group, for example 1- After reacting with a carbodiimide agent such as ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and activating, it reacts, for example, at 0 ° C. to room temperature for 1 to 16 hours to remove unbound linker molecules by membrane separation or the like. After that, a titanium oxide complex dispersed in an aqueous solvent with a water-soluble polymer is further mixed and reacted at, for example, 0 ° C. to room temperature for 1 to 16 hours to bind the antibody / linker molecule complex to the titanium oxide surface. And it can carry out by removing an unreacted antibody by membrane separation etc.

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

Example 1: Production of a titanium oxide composite bonded with polyethylene glycol 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 (manufactured by GE Healthcare 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 subjected to ultrasonic treatment at 200 kHz for 30 minutes using an ultrasonic generator MIDSONIC 200 (manufactured by Kaijo). 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 using a Zetasizer Nano ZS (manufactured by Sysmex), Various parameters of the solvent were set to the same values as water, and the measurement was performed at 25 ° C. As a result, the dispersed particle size was 36.4 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: manufactured by 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 prepare a dispersion of a titanium oxide complex bound with polyethylene glycol. 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 a titanium oxide complex bonded with polyethylene glycol, and various parameters of the solvent were set to the same values as water and measured at 25 ° C. As a result of cumulant analysis, the dispersed particle size was 54.2 nm.

Example 2: Production of titanium oxide composite bonded with polyacrylic acid In the same manner as in Example 1, an acidic titanium oxide sol having a solid component of 20% was finally prepared.

  After 0.6 ml of this acidic titanium oxide sol was adjusted to 20 ml with dimethylformamide (DMF) and dispersed, 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) was dissolved was added and stirred. And mixed. The solution was transferred to a hydrothermal reaction vessel HU-50 (manufactured by Sanai Kagaku) 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 precipitate surface was washed with ethanol, and then 1.5 ml of water was added to obtain a dispersion of a titanium oxide complex bonded with polyacrylic acid. 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 using a Zeta Sizer Nano ZS, charged with 0.75 ml of a dispersion of a titanium oxide complex bonded with polyacrylic acid in a zeta potential measurement cell, and various parameters of the solvent were set to the same values as water, and 25 ° C. I went there. As a result, the dispersed particle size was 53.6 nm, and the zeta potential was −45.08 mV.

Example 3 Production of Titanium Oxide Composite Bonded with Polyethyleneimine Similar to Example 1, an acidic titanium oxide sol having a solid content of 20% was finally prepared.

  3 ml of the obtained titanium oxide sol was dispersed in 20 ml of dimethylformamide (DMF), 10 ml of DMF in which 450 mg of polyethyleneimine (manufactured by Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 10,000 was dissolved was added, and the mixture was stirred and mixed. The solution was transferred to a hydrothermal reaction vessel HU-50 (manufactured by Sanai Kagaku) 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 precipitate surface was washed with ethanol, and then 1.5 ml of water was added to obtain a dispersion of a titanium oxide complex bonded with polyethyleneimine. 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 using a Zetasizer Nano ZS, and a 0.75 ml dispersion of a titanium oxide complex in which polyethylene imine was bound to a zeta potential measurement cell was charged, and various parameters of the solvent were set to the same values as water, and the temperature was adjusted to 25 ° C. I went. As a result, the dispersed particle size was 57.5 nm, and the zeta potential was 47.5 mV.

Example 4: Binding of dihydroxyphenylpropionic acid to titanium oxide composite Using the titanium oxide composite obtained in Example 1 and dihydroxyphenylpropionic acid, they were mixed in ultrapure water with the composition shown in Table 1, and the total Prepared to 1 ml. Titanium oxide composites A to C were used in the respective compositions.

The prepared solution was allowed to stand at room temperature for 4 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 ℃
From the obtained amount of change, the amount of dihydroxyphenylpropionic acid bonded per mass of titanium oxide particles was the result shown in Table 2.

  Furthermore, unreacted dihydroxyphenylpropionic acid was removed from 1 ml of this solution with 1.5 ml of water by using a natural fall column NAP-10 (manufactured by GE Healthcare Bioscience) for buffer exchange. 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 titanium oxide composites (titanium oxide composites A to C) bonded with dihydroxyphenylpropionic acid was confirmed.

Example 5: Binding of anti-EpCAM antibody to titanium oxide complex bound with dihydroxyphenylpropionic acid Among the titanium oxide complexes bound with dihydroxyphenylpropionic acid obtained in Example 4, a solution of titanium oxide complex B and 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Dojin Chemical) 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-anti-human EpCAM monoclonal antibody (mouse IgG: manufactured by Biomatrix Laboratories) prepared with the same buffer was added to a concentration of 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. From the size of the separated molecules, the flow-through fraction was considered to be a solution containing a titanium oxide-antibody complex bound with antibody molecules. As for the fraction in which the anti-human EpCAM 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, the titanium oxide-antibody complex in which the titanium oxide complex bound to dihydroxyphenylpropionic acid is bound to an antibody molecule that specifically binds to the surface antigen of bladder cancer cells via dihydroxyphenylpropionic acid. It was confirmed that the body could be produced.

Example 6: Binding of fluorescent dye to titanium oxide-antibody complex The titanium oxide-antibody complex obtained in Example 5 was 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 the dispersion were mixed at 1: 9 to make 1 ml, and a binding reaction was performed at room temperature for 4 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, 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 4.0 × 10 ⁻⁵ dopamine-g / titanium oxide particles-g per mass of titanium oxide particles. From this, the linker molecule as a whole was 9.0 × 10 ⁻⁵ linker molecule-mol / titanium oxide particle-g per mass of titanium oxide particles.

  Further, 1.0 ml of this solution was recovered with 1.5 ml of water using a natural fall column for buffer exchange NAP-10 (manufactured by GE Healthcare Bioscience) to remove unreacted dopamine. 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 a titanium oxide-antibody complex bound with dopamine was confirmed. Next, this titanium oxide-antibody complex to which dopamine was bound was 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 a natural drop column PD-10 for buffer exchange (manufactured by GE Healthcare Bioscience) with 3.5 ml of water. 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 confirmed to exhibit fluorescence at an excitation wavelength of 555 nm and a fluorescence wavelength of 575 nm. From these facts, production of a fluorescent molecule-bound titanium oxide-antibody complex in which a fluorescent molecule was bound to dopamine-bound titanium oxide-antibody complex via dopamine was confirmed.

Example 7: Evaluation of dispersibility of titanium oxide composite The titanium oxide composite obtained in Example 1 (referred to as titanium oxide composite D) and the titanium oxide composites A to C obtained in Example 4 were each phosphate buffered. It added so that it might become 0.05% of solid component with respect to the physiological saline, and it left still at room temperature for 1 hour. Thereafter, in the same manner as in Example 1, the dispersed particle size and the zeta potential were measured by a dynamic light scattering method using Zeta Sizer Nano ZS. The results are shown in Table 3. In the titanium oxide composites A to D, it was confirmed that there was no significant change in the dispersed particle size and the zeta potential.

Example 8: Dispersibility evaluation of titanium oxide-antibody complex The titanium oxide-antibody complex obtained in Example 5 was added to a phosphate buffered saline so that the solid component was 0.05%. The mixture was allowed to stand at room temperature for 1 hour. Thereafter, in the same manner as in Example 1, the dispersed particle size and the zeta potential were measured by a dynamic light scattering method using Zeta Sizer Nano ZS. As a result, the dispersed particle size was 50.4 nm and the zeta potential was −7.19 mV, confirming that there was no significant change compared to the result of Example 7.

Example 9: Evaluation of singlet oxygen generation ability of titanium oxide composite by ultrasonic irradiation Titanium oxide composite obtained in Example 1 (referred to as titanium oxide composite D) and titanium oxide composite A obtained in Example 4 -C was prepared so that it might become 0.05% of solid component with respect to each phosphate buffered saline. In addition, only phosphate buffered saline was prepared as a control. Single Oxygen Sensor Green Reagent (Molecular Probes), a reagent for measuring the production of singlet oxygen, was mixed with 3 ml of each solution according to the manual to prepare a test solution. Using an ultrasonic irradiation device (manufactured by OG Giken, ULTRASONIC APPARATUS ES-2: 1 MHz), an ultrasonic wave was irradiated for 3 minutes at 0.4 W / cm 2 in a 50% duty cycle operation, and 400 μl of a solution before and after irradiation was used as a measurement sample. Collected one by one. For each measurement sample, the fluorescence intensity at Ex = 488 nm and Em = 525 nm due to singlet oxygen generation was measured with a fluorescence spectrophotometer (RF-5300PC; manufactured by Shimadzu Corporation). The result was as shown in FIG. As shown in FIG. 2, it was confirmed that the titanium oxide composites A to D generate singlet oxygen more efficiently by ultrasonic irradiation as compared with the control. Further, it was considered that the production of singlet oxygen was suppressed as the amount of linker bonded per mass of titanium oxide particles was increased.

Example 10: Evaluation of antigen binding of titanium oxide-antibody complex For confirmation of binding to an antigen by an SPR sensor, a sensor chip C1 (manufactured by BIAcore) is set in an SPR sensor device (BIACORE1000, manufactured by BIAcore), and EpCAM according to the manufacturer's manual -Immobilization reaction of Fc chimeric protein (R & D SYSTEMS) was performed. 50 μl NHS-EDC mixed solution contained in BIAcore amine coupling kit (manufactured by BIAcore) was flowed at 5 μl / min to succinylate carboxyl groups on the surface of the sensor chip, and then 10 mmol / l acetate-sodium acetate buffer solution (pH 5. The reaction was carried out by loading 50 μl of the EpCAM-Fc chimeric protein solution dissolved in 1) to 1 g / l at a flow rate of 5 μl / min. After the reaction, 50 mol of 1 mol / l ethanolamine contained in the BIAcore amine coupling kit was loaded at a flow rate of 5 μl / min to block a succinyl group that was not involved in the binding. This resulted in approximately 0.8 ng / mm ² of EpCAM-Fc chimeric protein binding. Next, a dispersion of the titanium oxide-antibody complex obtained in Example 5 prepared at 0.05 (wt / vol)% was loaded at 60 μl at a flow rate of 30 μl / min. After confirming the reaction of the sensorgram, 30 μl of 100 mmol / l glycine-NaOH buffer solution (pH 12.0) was loaded at 30 μl / min, and the dissociation reaction from the sensor was performed. Sensorgram analysis was performed using Biomolecular Interaction Analysis (BIA) evaluation software (version 3.5, manufactured by BIAcore). The result obtained by similarly loading the titanium oxide composite obtained in Example 1 as a background was subtracted. The results were as shown in FIG. These results indicate that the titanium oxide-antibody complex binds strongly to the antigen.

Example 11: Binding of dihydroxyphenylpropionic acid to a titanium oxide complex 2
Using the titanium oxide complex and dihydroxyphenylpropionic acid obtained in Example 1, 1) 20 mmol / l acetic acid-sodium acetate buffer solution (pH = 3.6), 2) 20 mmol / l MES buffer solution ( Manufactured by Dojindo; pH = 6.0), 3) In a 20 mmol / l HEPES buffer solution (produced by Dojindo; pH = 8.1), the final concentration of titanium oxide complex was 2%, and dihydroxyphenylpropionic acid was added. The final concentration was 50 mmol / l to prepare a total of 0.8 ml.

  The prepared solution was stirred at 40 ° C. for 25 hours. The absorption spectrum in the ultraviolet-visible light region (200-600 nm) of each solution was confirmed by an ultraviolet-visible light spectrophotometer. Regarding the solution in which only dihydroxyphenylpropionic acid was mixed, in 1) 20 mmol / l acetic acid-sodium acetate buffer solution (pH = 3.6), there was almost no change compared with 0 hour after adjustment, 2) In 20 mmol / l MES buffer solution (pH = 6.0) and 3) 20 mmol / l HEPES buffer solution (pH = 8.1), changes in absorption spectrum were confirmed compared to 0 hours after adjustment. It was confirmed by visual observation that the color changed to light red. From these results, it was considered that dihydroxyphenylpropionic acid is unstable at pH = 6.0 or higher. Moreover, about the solution which mixed the titanium oxide complex and dihydroxyphenylpropionic acid, in 1) 20 mmol / l acetic acid-sodium acetate buffer solution (pH = 3.6), compared with 0 hours after adjustment, the absorption spectrum of The change was confirmed, and it was confirmed by visual observation that the color changed to dark brown. Since there was no significant change in dihydroxyphenylpropionic acid alone, this change was thought to be due to the binding of dihydroxyphenylpropionic acid to the titanium oxide complex and charge transfer.

Next, 1) In the 20 mmol / l acetic acid-sodium acetate buffer solution (pH = 3.6), the solution after stirring for 0 hour and 25 hours after adjustment was subjected to capillary electrophoresis under the following conditions. The amount of change in dihydroxyphenylpropionic acid was determined by confirming a peak at an absorption wavelength of 214 nm of the 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 borate buffer solution (pH 9.0)
・ Voltage: 25kV
・ Temperature: 20 ℃
From the obtained amount of change, 1) the amount of dihydroxyphenylpropionic acid bound per mass of titanium oxide particles in a 20 mmol / l acetic acid-sodium acetate buffer solution (pH = 3.6) was 7.7 × 10 ⁻⁴ dihydroxyphenylpropiate. On-acid-mol / titanium oxide particles-g.

Example 12: Cell killing test using ultrasonic irradiation The titanium oxide-antibody complex obtained in Example 5 was added to 3 ml of RPMI 1640 medium (manufactured by Invitrogen) containing 10% serum containing 1 × 10 ⁵ cells / ml of Jurkat cells. The test solution was adjusted to a final concentration of 0.05% and 0%. Each test solution was subjected to ultrasonic irradiation (0.5 W / cm ² , 50% pulse) for 15 seconds using an ultrasonic irradiation device (ULTRASONIC APPARATUS ES-2: 1 MHz, manufactured by OG Giken Co., Ltd.). The number of cells was measured according to the manufacturer's instructions using the MTT assay (manufactured by Dojin Chemical), and the cell viability in each test solution was calculated with the number of cells before irradiation as 100%. As a result, the cell viability was 78.7% at a final concentration of 0.05%, and the cell viability was 90.3% at a final concentration of 0%. From these, the cell killing effect by ultrasonic irradiation of the titanium oxide-antibody complex was confirmed.

Example 13: Binding of ferrocenecarboxylic acid and dopamine to titanium oxide complex 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. Manufactured). Similarly, using DMF, 200 mM Benzotriazole-1-yl-oxy-trispyrrolophosphonium hexafluorophosphate (PyBop; manufactured by Merck), 200 mM 1-hydroxybenzotriazole (HoBt; manufactured by Dojindo), 20 mM N ethylamine, 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 obtained in Example 1 was adjusted to 1% solid component with ultra pure 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, preparation of a complex-bound titanium oxide complex of ferrocenecarboxylic acid and dopamine hydrochloride was confirmed.

Example 14: Binding of ferrocenecarboxylic acid and dopamine to titanium oxide-antibody complex In Example 13, instead of the titanium oxide complex obtained in Example 1, the titanium oxide-antibody complex obtained in Example 5 was used. Except for the above, a complex-bound titanium oxide-antibody complex of ferrocenecarboxylic acid and dopamine hydrochloride was prepared in the same manner.

Example 15: Evaluation of hydroxy radical-forming ability of ferrocenecarboxylic acid and dopamine hydrochloride complex-bound titanium oxide complex by ultrasonic irradiation Complex-bound titanium oxide complex of ferrocenecarboxylic acid and dopamine hydrochloride obtained in Example 13 (oxidation) Titanium complex E) was prepared such that the solid component was 0.05% with respect to phosphate buffered saline (pH 7.4). Further, only phosphate buffered saline (pH 7.4) was used as a control. 3 ml of each solution was prepared and used as a test solution. Ultrasonic irradiation (0.4 W / cm 2, 50% pulse) is performed for 3 minutes using an ultrasonic irradiation device (manufactured by OG Giken, ULTRASONIC APPARATUS ES-2: 1 MHz). A reagent for measuring the production of hydroxyphenylfluorescein (HPF, manufactured by Daiichi Chemicals) is mixed according to the manual, and allowed to stand at room temperature for 15 minutes and 30 minutes. 400 μl of the solution before and after irradiation as a measurement sample at each standing time Collected. For each measurement sample, the fluorescence intensity at Ex = 490 nm and Em = 515 nm due to the generation of hydroxy radicals was measured with a fluorescence spectrophotometer (RF-5300PC; manufactured by Shimadzu Corporation). The result was as shown in FIG. As shown in FIG. 4, it was confirmed that the titanium oxide complex E efficiently generates hydroxy radicals by ultrasonic irradiation as compared with the control. In addition, it was considered that the titanium oxide complex E generates hydroxy radicals continuously because the fluorescence value increases with the standing time.

Example 16: Binding of antibody to titanium oxide complex via dopamine 0.1 mg of anti-human EpCAM monoclonal antibody (mouse IgG: manufactured by Biomatrix Laboratories) Coupling buffer (pH 5.5; Catalog No. 153-6054, Bio And 25 ml / 1.2 ml of sodium periodate aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto and reacted at room temperature for 1 hour, followed by ultrapure water. Was added to a 2.5 ml solution, and 3.2 ml of a 20 mM MES buffer solution (pH 5.5) was recovered using a natural drop column PD-10 (manufactured by GE Healthcare Bioscience) for buffer exchange. Removal of sodium periodate from the reaction, Amicon Ultra-15 (MWCO = 5000; manufactured by Millipore) ) By centrifugation (1500 g, 15 min) and concentrated to 0.7 ml to obtain an oxidized antibody solution. In addition, 0.5 ml of 200 mM aqueous dopamine hydrochloride (manufactured by Wako Pure Chemical Industries) and 0.1 ml of 70 mM succinimidyl-4-hydrazinonicotinate-acetone hydrazone (SANH; manufactured by PIERCE) aqueous solution were mixed, and a 100 mM HEPES buffer solution ( The total amount was adjusted to 1 ml with pH 8.1) and ultrapure water. After reacting at room temperature for 1 hour, the binding of SANH and dopamine was confirmed by thin layer chromatography to obtain a complex solution of SANH and dopamine. These 0.7 ml of the oxidized antibody solution, 0.1 ml of the complex solution of SANH and dopamine and 0.2 ml of Coupling buffer (pH 5.5; Catalog No. 153-6054, manufactured by Bio-Rad) were mixed and mixed at 4 ° C. After reaction for 16 hours, elute with PBS buffer solution (phosphate saline) using PD-10 (GE Healthcare Bioscience), a natural buffer column for buffer exchange, and complex of antibody, SANH and dopamine Then, the unreacted complex of SANH and dopamine was removed. Thus, a complex of the antibody, SANH and dopamine was obtained. Next, 2 ml of the 5 (wt / vol)% titanium oxide complex obtained in Example 1 and 1 ml of the antibody / SANH / dopamine complex were mixed and reacted at 4 ° C. for 16 hours. mm PBCC membrane (MWCO = 300000; Catalog No. PBMK04310, manufactured by Millipore) and stirred cell Model 8050 (Catalog No. 5122, manufactured by Millipore), unbound with a 334 ml solution change at 10 psi according to the manufacturer's protocol The complex of antibody, SANH and dopamine was removed. In this way, a dispersion of titanium oxide-anti-EpCAM antibody complex particles was obtained.

  Next, a titanium oxide-anti-AFP antibody was exactly the same except that an anti-alpha fetoprotein (anti-AFP) antibody (mouse IgG: NB013, manufactured by Japan Biotest Laboratories) was used instead of the anti-human EpCAM monoclonal antibody. A dispersion of composite particles was obtained.

Example 17: Evaluation of antigen binding of titanium oxide-antibody complex For confirmation of binding to an antigen by an SPR sensor, a sensor chip C1 (manufactured by BIAcore) was set in an SPR sensor device (BIACORE1000, manufactured by BIAcore), and EpCAM according to the manufacturer's manual -Immobilization reaction of Fc chimeric protein (R & D SYSTEMS) was performed. A mixed solution of phosphate buffer (10 mM phosphate buffer (pH 7.4), 150 mM NaCl, 0.05 (wt / vol)% Tween 20) was used as the mobile phase. After 200 μl of NHS-EDC mixed solution contained in BIAcore amine coupling kit (manufactured by BIAcore) was flowed at 30 μl / min to succinylate carboxyl groups on the surface of the sensor chip, 90 mmol / l acetate-sodium acetate buffer solution (pH 5. The reaction was carried out by loading 180 μl of EpCAM-Fc chimeric protein solution dissolved at 100 μg / ml in 0) at a flow rate of 30 μl / min. After the reaction, 1 mol / l ethanolamine contained in the BIAcore amine coupling kit was loaded at 150 μl and a flow rate of 30 μl / min to block succinyl groups that were not involved in binding. As a result, a response of about 500 RU was obtained. A dispersion of 0.01 (wt / vol)% titanium oxide-anti-EpCAM antibody complex particles prepared in Example 16 and titanium oxide-anti-AFP antibody complex particles were loaded at 90 μl at a flow rate of 30 μl / min. After confirming the reaction of the sensorgram, 100 μl of 100 mmol / l glycine-NaOH buffer solution (pH 12.0) was loaded at 30 μl / min, and the dissociation reaction from the sensor was performed. Sensorgram analysis was performed using Biomolecular Interaction Analysis (BIA) evaluation software (version 3.5, manufactured by BIAcore). The result obtained by similarly loading the titanium oxide composite obtained in Example 1 as a background was subtracted. As a result, a response of 150 RU was obtained for the titanium oxide-anti-EpCAM antibody composite particles, and a response of 3 RU was obtained for the titanium oxide-anti-AFP antibody composite particles. From these results, it was shown that the titanium oxide-anti-EpCAM antibody complex particles bound strongly to the antigen. Further, the titanium oxide-anti-AFP antibody complex particles did not bind to the antigen.

Example 18: Evaluation of antigen binding of titanium oxide-antibody complex (FACS)
In order to confirm the binding of the titanium oxide-antibody complex to bladder cancer cells, evaluation was performed using a flow cytomelloy (FACS) method. First, logarithmically growing human bladder cancer cell line RT112 was mixed with PBS + BSA + EDTA (PBS buffer (phosphate physiological saline) +0.5 (wt / vol)% bovine serum albumin (Invitrogen)) + 2 mmol / l ethylenediaminetetraacetic acid (sum) The number of cells was adjusted to 2 × 10 ⁵ cells by Kojunkaku Kogyo Co., Ltd.), dispensed into a 5 ml tube, and centrifuged (1500 g, 5 min, 4 ° C.) to remove the supernatant. Thereto was added 1 ml of a dispersion of the titanium oxide-anti-EpCAM antibody complex particles prepared in Example 16 and made 0.1 (wt / vol)% using PBS buffer (phosphate physiological saline). After suspension, the mixture was allowed to stand on ice for 30 minutes. Next, 3 ml of PBS + BSA + EDTA mixed solution was added for washing, and the supernatant was removed by centrifugation (1500 g, 5 min, 4 ° C.). As a secondary antibody, 1 μl of Biotinylated anti-mouse IgG (H + L) (manufactured by Vector) was added, and left on ice for 30 min. Next, 3 ml of PBS + BSA + EDTA mixed solution was added for washing, and the supernatant was removed by centrifugation (1500 g, 5 min, 4 ° C.). For labeling, 1 μl of Strept Avidine Alexa Fluore-647 conjugate (manufactured by Invitrogen) was added and allowed to stand on ice for 30 min. Next, 3 ml of PBS + BSA + EDTA mixed solution was added for washing, and the supernatant was removed by centrifugation (1500 g, 5 min, 4 ° C.). This washing was repeated twice. Then, 200 μl of PBS + BSA + EDTA mixed solution was added and suspended to obtain test solution A.

Also, logarithmically growing human bladder cancer cell line RT112 was adjusted to a PBS + BSA + EDTA mixed solution (with a cell number of 2 × 10 ⁵ cells), dispensed into a 5 ml tube, and centrifuged (1500 g, 5 min, 4 ° C.) to obtain a supernatant. After suspending 1 ml of an anti-human EpCAM monoclonal antibody (mouse IgG: manufactured by Biomatrix Laboratories) adjusted to 0.1 mg / ml with PBS buffer (phosphate physiological saline), Then, 3 ml of PBS + BSA + EDTA mixed solution was added for washing, and the supernatant was removed by centrifugation (1500 g, 5 min, 4 ° C.) Zenon Alexa Fluore-647 Mouse IgG1 Labeling for labeling kit (manufactured by Invitrogen) The solution was labeled according to Col, and finally suspended in 200 μl of a PBS + BSA + EDTA mixed solution to obtain Test Solution B.

Controls prepared by suspending the test solution A, test solution B and the logarithmically growing human bladder cancer cell line RT112 in 2 × 10 ⁵ cells in 200 μl of PBS + BSA + EDTA mixed solution were prepared, respectively. FACScalibur flow site Detection was performed using a meter (manufactured by Becton Dickinson) according to the manufacturer's protocol, with settings of excitation laser 633 nm and fluorescence detection filter 650-670 nm. The result is shown in FIG. A cell group with high fluorescence intensity was not confirmed in the control. On the other hand, in the test solution A and the test solution B, a cell group having high fluorescence intensity was confirmed. These results indicate that the titanium oxide-anti-EpCAM antibody complex particles bind strongly to bladder cancer cells having antigens.

Claims (16)

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. A titanium oxide composite,
A linker molecule bonded to the surface of the titanium oxide complex;
A bladder that is a titanium oxide-antibody complex comprising an antibody that specifically binds to a surface antigen of bladder cancer cells, which is bound via the linker molecule, and has catalytic activity by irradiation with ultrasonic waves A cancer therapeutic agent,
The linker 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- or 6-membered heterocyclic group having or not having a substituent, or c) substitution A compound comprising a saturated or unsaturated 5- or 6-membered cyclic hydrocarbon group with or without a group,
Bonded with the titanium oxide via the functional group, without polymerizing the functional groups,
The bladder cancer therapeutic agent is injected into the bladder and combined with the surface antigen of bladder cancer cells, which is an antigen, and administered by removing unbound bladder cancer therapeutic agent by urination or intravesical washing,
A therapeutic agent for bladder cancer, which is used in a method of subsequently irradiating a bladder with ultrasonic waves. 2. The bladder cancer treatment according to claim 1, wherein the amount of the linker molecule bound is 1 × 10 ⁻⁶ to 1 × 10 ⁻³ mol / titanium oxide particles-g per mass of the titanium oxide particles. Agent.   The therapeutic agent for bladder cancer according to claim 1 or 2, wherein the linker molecule is a catechol.   The bladder cancer therapeutic agent according to claim 3, wherein the linker molecule is at least one selected from the group consisting of dopamine and dihydroxyphenylpropionic acid.   The therapeutic agent for bladder cancer according to any one of claims 1 to 4, wherein the water-soluble polymer has a weight average molecular weight of 5000 to 40000.   The therapeutic agent for bladder cancer according to any one of claims 1 to 5, wherein the water-soluble polymer comprises at least one selected from the group consisting of polyethylene glycol, polyacrylic acid, and polyethyleneimine.   The bladder cancer therapeutic agent according to any one of claims 1 to 6, which has a particle size of 20 to 200 nm.   The bladder cancer therapeutic agent according to any one of claims 1 to 7, wherein the antibody is selected from the group consisting of an anti-EpCAM antibody, an anti-CD44 antibody, and an anti-EGF receptor antibody.   The therapeutic agent for bladder cancer according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody.   The therapeutic agent for bladder cancer according to any one of claims 1 to 9, wherein a fluorescent molecule is further bound via the linker molecule.   The therapeutic agent for bladder cancer according to any one of claims 1 to 10, further comprising a molecule containing a low-valent transition metal bonded through the linker molecule.   A dispersion comprising the bladder cancer therapeutic agent according to any one of claims 1 to 11 and a solvent in which the bladder cancer therapeutic agent is dispersed.   The dispersion according to claim 12, wherein the solvent is an aqueous solvent.   The dispersion according to claim 12 or 13, wherein the solvent has a pH of 5 to 8.   The dispersion according to any one of claims 12 to 14, wherein the solvent is physiological saline.   The dispersion liquid as described in any one of Claims 12-15 in which the said bladder cancer therapeutic agent contains 0.001-1 mass%.

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