JP2005314409A - Dispersion containing titanium dioxide complex having molecular identifying ability - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare a dispersion containing a titanium dioxide complex specifically binding to an endocrine disruptor, an etiological molecule, a cancer cell, or the like, carrying out degradation thereof by photocatalytic actions and having a molecular identifying ability. <P>SOLUTION: This dispersion containing the titanium dioxide complex having the molecular identifying ability is obtained as follows. The surfaces of titanium dioxide fine particles are modified with a hydrophilic polymer in which carboxy groups are bound to titanium dioxide with ester bonds. Molecules having a specific binding ability to an objective molecule are fixed onto the carboxy residues of the hydrophilic polymer and the resultant complex is dispersed in an aqueous solution. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention immobilizes endocrine disrupting substances or molecules having specific binding ability to pathogenic molecules, cancer cells, etc., and shows the molecular action of degrading these substances, molecules, cells by irradiation with ultraviolet rays, etc. The present invention relates to a titanium dioxide composite having a function.

  In recent years, a material in which a biomolecule such as DNA having molecular discrimination ability of an endocrine disrupting substance is immobilized on a support and thereby imparted selective binding has been proposed as an environmental purification material (see, for example, Patent Document 1). ). Anatase-type titanium dioxide has a photocatalytic action and is known to decompose organic substances such as microorganisms, dirt, and malodorous substances by its strong oxidizing power. In particular, it is expected to be effective for environmental purification because it exhibits a strong decomposing action even on a hardly decomposable substance such as an endocrine disrupting substance (see, for example, Non-Patent Document 1). Furthermore, the present invention has been devised to increase the decomposition efficiency of titanium dioxide by combining titanium dioxide and an inorganic adsorbent such as activated carbon or zeolite (for example, see Patent Document 2). Also in the surface treatment of titanium dioxide, it has been devised to promote the oxidation and reduction reaction of the photocatalyst by precipitating a reduction reaction promoting catalyst metal such as palladium on the surface of the photocatalyst such as titanium dioxide (for example, Patent Document 3). reference).

  However, the selective binding material for the endocrine disrupting substance such as DNA does not have a reliable means for removing or decomposing the bound endocrine disrupting substance, and the purification capacity is limited due to the problem of adsorption saturation. In addition, the idea to increase the ability of titanium dioxide as a photocatalyst is not directed to the binding or decomposition of specific substances. Therefore, for example, it was impossible to selectively bind and decompose only with an endocrine disrupting substance. In this way, in the field of environmental purification, only the target substance is identified and selectively combined, and this is decomposed by the strong oxidizing power of the photocatalyst, that is, the “combination of molecular discriminating ability and photocatalytic ability” technology using titanium dioxide. Is not known.

  On the other hand, as a new drug administration form in the medical field, a system (drug delivery system: DDS) designed to gradually release a drug over time in the body or body surface has been attracting attention. This is intended to maximize the efficacy of existing drugs and to control their side effects to a minimum. Examples of the drug carrier in DDS include non-degradable polymers and amino acid polymers (for example, see Patent Document 4), liposomes (for example, see Patent Document 5), and protein hollow nanoparticles (for example, see Patent Document 6). It has been actively studied. A further advancement of the concept of DDS is target-oriented (targeting) DDS. This is a system that delivers a drug to a required site and a required amount at a required time, and finally aims at a missile drug (missile therapy) that surely targets a lesion.

  In the case of a missile drug, targeting is performed by supporting a ligand on a DDS carrier and specifically recognizing and binding to a receptor present on the surface of a target cell. Examples of ligands corresponding to receptors that are targets for such active targeting include antigens, antibodies, peptides, glycolipids and glycoproteins. Among these, glycolipids of glycolipids and glycoproteins are important as information molecules in cell proliferation and differentiation, tissue development and morphogenesis, biological defense and fertilization mechanisms, or intercellular communication such as canceration and metastasis. In recent years, it has become clear that it plays a role (for example, refer nonpatent literature 2).

  Attempts have been made to apply titanium dioxide having strong photocatalytic resolution to such DDS (see Patent Document 7, Patent Document 8, and Non-Patent Document 3). This is intended to kill cancer cells by irradiating light such as ultraviolet rays after shooting metal particles such as gold carrying titanium dioxide into target cancer cells. Titanium dioxide is known to be a very stable substance in the air and in solution, and is safe without toxicity in the animal body (shielded from light). In addition, since the activation of titanium dioxide can be controlled by turning on / off light, application to DDS for cancer treatment is expected.

  Furthermore, a titanium dioxide composite that combines these titanium dioxide and a polymer base material, which is suitable as a medical material, has been proposed. For example, there is a medical material in which a polymer base material and titanium dioxide are combined as a medical material for an artificial blood vessel or an artificial organ (see Patent Document 9). This is because by introducing a functional group capable of chemically bonding to titanium oxide into a highly biocompatible polymer substrate, the polymer substrate can be firmly bonded without being easily peeled off. Since it is not necessary to perform chemical pretreatment, there is no fear that the properties (physical properties) of titanium dioxide will be impaired or modified due to this chemical pretreatment, and it can be suitably used as a medical material. . In addition, there is a medical material in which titanium dioxide and a biomolecule are combined (see Patent Document 10). This is a photocatalyst of specific harmful substances such as viruses, bacteria, and toxins that may be mixed into blood by complexing titanium dioxide with biomolecules that specifically recognize and bind to specific substances. By being inactivated by function, it is useful as a technique that can efficiently separate blood components and blood protein component protein components without denaturation.

  However, the isoelectric point of titanium dioxide is around pH 6, and there is a problem that titanium dioxide particles aggregate under physiological conditions near neutrality. In these conventional manufacturing methods, the functionality of titanium oxide is limited to a membrane shape, a plate shape, or a particle shape that allows easy solid-liquid separation, so that titanium dioxide itself can be administered directly into blood vessels. It was impossible to use it as it was as a carrier for DDS.

  Furthermore, it is possible to bind to the surface of titanium dioxide while maintaining the activity of a molecule having a selective binding ability such as the ligand, and to maintain a high degree of dispersibility under physiological conditions near neutrality. No such technology is known, and the practical application of titanium dioxide as a DDS is currently difficult. That is, even in the medical field, the “combination of molecular discriminating ability and photocatalytic ability” technology using titanium dioxide has not yet been developed due to the above problems.

JP 2001-81098 A Japanese Patent Laid-Open No. 01-189322 Japanese Patent Laid-Open No. 60-14940 JP 09-255590 A JP 2003-226638 A JP 2003-286198 A JP 2002-316946 A JP 2002-316950 A JP 2004-143417 A JP 2003-116534 A Y. Ohko et al .: Environmental Science and Technology, 35, 2365-2368 (2001) N. Yamazaki et al .: Advanced Drug Delivery Review, 43, 225-244 (2000) R. Cai et al .: Cancer Research, 52, 2346-2348 (1992)

  The present invention has been made to solve the above-mentioned problem of “combination of molecular discrimination ability and photocatalytic ability” with titanium dioxide. That is, an object of the present invention is to provide a titanium dioxide complex having molecular discrimination ability, which specifically binds to endocrine disrupting substances, pathogenic molecules, cancer cells and the like and exhibits their degrading action by photocatalytic action. is there.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive studies, modified the surface of titanium dioxide fine particles with a hydrophilic polymer, and then further immobilized a molecule having a specific binding ability to the target molecule. The present inventors have found that a titanium complex can achieve both molecular discrimination ability and photocatalytic ability, and have completed the present invention.

  That is, in the titanium dioxide composite having molecular discrimination ability of the present invention, the surface of the titanium dioxide fine particles is modified with a hydrophilic polymer, and the carboxyl group of the hydrophilic polymer and titanium dioxide are bonded by an ester bond, A molecule having a specific binding ability to a target molecule is immobilized on a carboxyl residue of the hydrophilic polymer. By this method, it becomes possible to introduce a molecule having specific binding ability such as an antibody into titanium dioxide particles having photocatalytic action, and a titanium dioxide complex having molecular discrimination ability can be produced.

  The present invention provides a titanium dioxide complex having molecular discrimination ability, which has molecular discrimination ability against endocrine disrupting substances, pathogenic molecules, cancer cells and the like, and exhibits a decomposition reaction of these substances by photocatalysis. This complex has the ability to specifically identify and capture a target molecule in water or an aqueous solution and to strongly decompose it by ultraviolet irradiation or the like. In particular, the properties of this complex that can be used in an aqueous solvent, that can accurately identify and capture the target molecule, and that exhibit a strong photocatalytic activity are, for example, decomposition treatments of harmful substances such as aqueous endocrine disruptors. Or, it is extremely useful for application to medical fields such as destruction of specific pathogenic molecules and cancer cells.

Embodiments of the present invention will be specifically described with reference to the drawings. FIG. 1 is a schematic view showing a titanium dioxide complex having molecular discrimination ability of the present invention. That is, the titanium dioxide composite having molecular discrimination ability of the present invention is obtained by dispersing titanium dioxide fine particles (1) and a hydrophilic polymer (2) having a plurality of carboxyl groups in dimethylformamide at 90 to 180 ° C. After hydrothermal reaction for 12 hours to bond the two with an ester bond, the molecule (3) having a specific binding ability to the target molecule is immobilized on the carboxyl residue of the hydrophilic polymer (2). It is a thing. Here, the titanium dioxide and the hydrophilic polymer are ester-bonded because the titanium oxide on the particle surface is hydrated with water in the reaction system to form a hydroxyl group on the surface. This is due to the reaction with the carboxyl group to form an ester bond. Various analysis methods can be applied as a method for confirming the ester bond. For example, it can be confirmed by the presence or absence of infrared absorption in the vicinity of 1700 to 1800 cm ⁻¹ which is an ester bond absorption band by infrared spectroscopy. Moreover, the amino group which this molecule | numerator (3) has mainly is used for fixation | immobilization of the molecule | numerator (3) which has specific binding ability. It is possible to introduce an amino group by an appropriate modification method even for a molecule having no amino group, or to introduce a desired functional group or cross-linkage reactive with a carboxyl group other than the amino group. Is possible.

  The titanium dioxide fine particles (1) used in the present invention may have a dispersed particle size of 2 to 200 nm from the viewpoint of the degree of freedom of the form of use, for example, in the case of aggregation problems or application to the body for cancer treatment. desirable. Furthermore, as the titanium dioxide used in the present invention, both anatase type and rutile type can be suitably used. This is because the chemical properties of titanium dioxide with different crystal systems are almost the same, and with any crystal system, modification with water-soluble polymers and immobilization of molecules with specific binding ability It is because it is possible to do. For example, in order to destroy cancer cells, anatase type having a strong photocatalytic activity can be selected, and a desired crystalline titanium dioxide can be selected and used according to the application.

    Furthermore, if titanium dioxide is present on at least part of the particle surface, the properties of titanium dioxide on the particle surface are similar, for example, even a composite material of magnetic material and titanium dioxide. It is possible to immobilize a molecule having specific binding ability via a group. Therefore, even if it is a composite material of a magnetic material and titanium dioxide, it is possible to manufacture the titanium dioxide composite which has a molecular discriminating ability by the completely same method as the case of a single titanium dioxide particle.

  The hydrophilic polymer (2) used in the present invention is preferably a water-soluble polymer because it is assumed that the titanium dioxide composite is used in a state of being dispersed in an aqueous solution. As the water-soluble polymer used in the present invention, any polymer having a plurality of carboxyl groups can be applied. For example, carboxymethyl starch, carboxymethyl dextran, carboxymethyl cellulose, polycarboxylic acids, and carboxyl group units. And a copolymer (copolymer) having. 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. By modifying titanium dioxide with these hydrophilic polymers, it becomes possible to immobilize the molecule (3) having a desired specific binding ability to the carboxyl residue of the hydrophilic polymer. Furthermore, even after immobilization of the molecule (3), the titanium dioxide composite of the present invention can maintain a uniformly dispersed state in a wide pH range including near neutrality due to the electric repulsion between the remaining carboxyl groups.

  In the present invention, the molecule (3) having a specific binding ability that imparts molecular discrimination ability to the titanium dioxide complex is not limited to the following as long as it is a molecule that specifically binds to the target molecule. . A wide variety of such specific bonds between molecules have been revealed in vivo. Among these, protein is mentioned as the most important molecule. According to the present invention, antibodies, ligands, receptors, poly-oligopeptides, and even amino acids can be suitably immobilized as proteins. In addition, an amino group and a thiol group can be used for immobilization of a simple protein to a titanium dioxide complex, and an aldehyde group of a sugar can be used as a target functional group for immobilization in the case of a glycoprotein. In addition, biotin (or avidin) is introduced into the carboxyl group of titanium dioxide modified with a water-soluble polymer, and the protein is cross-linked with avidin (or biotin) to utilize the biotin: avidin interaction. It is also possible to fix it.

  Furthermore, the titanium dioxide composite of the present invention can present specific factors and ligands on the particle surface. Thus, the complex can be introduced into a cell by specific ligand: receptor binding to a cell such as a cancer cell expressing a specific receptor, for example. These factors and ligands include growth factors such as epidermal growth factor (EGF), transforming growth factor, platelet-derived growth factor, bone morphogenetic factor, and nerve growth factor, as well as interferon, interleukin, Examples include hormones and ligands such as colony stimulating factor, tumor necrosis factor, erythropoietin, Fas antigen, and activin. These proteins can also be immobilized in the same manner as described above. That is, it is possible to construct a targetable missile drug for a specific cell that specifically expresses a receptor corresponding to these.

  In recent years, nucleic acid aptamers that specifically bind to specific proteins have attracted attention. Such an aptamer can also be used as the molecule (3) having specific binding property that imparts the molecular discrimination ability of the present invention. When nucleic acid is immobilized, the DNA is amplified by polymerase chain reaction (PCR), and then modified by synthesizing modified DNA using an aminated primer, biotinylated primer, and thiolated primer. It can be immobilized on titanium dioxide. For example, when aminated DNA is used for immobilization, an ester such as N-hydroxysuccinimide (NHS) is previously introduced into the carboxyl group of the modified titanium dioxide, and the aminated DNA is converted into the modified titanium dioxide by a nucleophilic substitution reaction. It can be covalently bonded. In the case of using thiolated DNA, it is also possible to immobilize thiolated DNA on modified titanium dioxide by reacting NHS with a carboxyl group and then reacting with 2- (2-pyridinyldithio) ethanamine. .

  When the aldehyde group of the molecule to be immobilized is used, NHS is reacted with the carboxyl group, and then the molecule to be immobilized is bound to the modified titanium dioxide by using hydrazine and reduced with sodium cyanoborohydride. In addition, if the carboxyl group is biotinylated using biotin hydrazide or aminated biotin, an avidin-immobilized molecule can be easily introduced onto the modified titanium dioxide. Thus, by appropriately selecting reagents, modification and cross-linking methods, it is possible to easily immobilize molecules (3) having various specific binding abilities to carboxyl residues introduced on the modified titanium dioxide. Is possible.

  As described above, if the functional group capable of binding to the carboxyl residue introduced on the modified titanium dioxide is present and the binding technique is clear, the molecule (3) having a specific binding ability can be a protein or nucleic acid. Alternatively, in addition to saccharides, lipids and various physiologically active substances can be suitably used.

  On the other hand, the titanium dioxide complex having molecular discrimination ability of the present invention is uniformly dispersed in a neutral aqueous solvent due to physiological conditions in the living body when applying to aqueous harmful substance treatment, medicine or medical treatment. It is required that The titanium dioxide composite in the dispersion liquid preferably has a dispersed particle size of 2 to 500 nm from the viewpoint of dispersibility. Furthermore, in the case of application to the body for cancer treatment, the dispersed particle size is more preferably 50 to 200 nm from the viewpoint of the effect of accumulation in cancer cells. By setting it as such a range, high dispersion | distribution of the grade which can be couple | bonded with the activity of the molecule | numerator which has a selective binding ability on the titanium dioxide surface over 24 hours or more on physiological conditions is attained. As described above, since the titanium dioxide complex having molecular discrimination ability of the present invention has a residual carboxyl residue, repulsive force derived from the negative charge of the carboxyl group acts between the complexes in an aqueous solvent. . Therefore, even in an aqueous solution in a wide pH range of pH 3 to 13, this complex can maintain a uniformly dispersed state without aggregation. Therefore, it is possible to provide a uniform and stable dispersion in which the titanium dioxide complex having molecular discrimination ability of the present invention is dispersed in water, various pH buffer solutions, infusion solutions, or physiological saline. In addition, ointments and sprays containing this dispersion can also be produced. This characteristic is extremely useful particularly when titanium dioxide is applied to DDS inside and outside the body. That is, since the dispersion of the titanium dioxide complex having molecular discrimination ability of the present invention does not aggregate even under physiological conditions near neutrality, targeting is performed by direct injection into the affected tissue or intravenous injection. It becomes possible. In addition, an ointment or spray containing this dispersion can be applied directly to the affected area such as the skin, and phototherapy can be performed with sunlight or an ultraviolet lamp.

  Furthermore, it goes without saying that the titanium dioxide composite having molecular discrimination ability of the present invention can be used alone as a DDS, and can be used as a form of DDS in other carriers. Although there is no restriction | limiting in particular as a carrier in this case, A liposome, a virus particle, a hollow nanoparticle, etc. can be used suitably.

  The light source device for exciting and activating the titanium dioxide complex having molecular discrimination ability of the present invention does not need to be special, but the wavelength is desirably 400 nm or less because of the band gap of titanium dioxide. For external use such as skin, sunlight, a normal ultraviolet lamp, or black light can be suitably used. Moreover, what is necessary is just to irradiate an ultraviolet-ray by attaching an ultraviolet fiber to an endoscope with respect to the affected part in a body. Furthermore, in the case of assuming phototherapy in which the affected area is destroyed by locally irradiating the affected area with ultraviolet rays around 280 nm, the titanium dioxide complex having the molecular discrimination ability of the present invention is applied as its action enhancing agent. It is also possible to do.

  Hereinafter, the present invention will be described in detail according to examples. However, the present invention is not limited to this embodiment.

(Example 1)
Introduction of polyacrylic acid into titanium dioxide 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 stirred at 80 ° C. for 8 hours for peptization. After completion of peptization, the mixture was filtered through a 0.45 μm filter, and the solution was exchanged using a desalting column (PD10; Amersham Pharmacia Bioscience) to prepare anatase-type titanium dioxide sol having a solid content of 1%. This dispersion was placed in a 100 ml vial and sonicated at 200 Hz for 30 minutes. The average dispersed particle sizes before and after the ultrasonic treatment were 36.4 nm and 20.2 nm, respectively. After sonication, the solution was concentrated to prepare a 20% solid component titanium dioxide sol (anatase type).
0.75 ml of the obtained titanium dioxide sol was dispersed in 20 ml of dimethylformamide (DMF), and after adding 10 ml of DMF in which 0.2 g of polyacrylic acid (average molecular weight: 5000, Wako Pure Chemical Industries) was dissolved, the mixture was stirred and mixed. . The solution was transferred to a hydrothermal reaction vessel, and hydrothermal synthesis was performed at 180 ° C. for 6 hours. After completion of the reaction, the reaction vessel was cooled to 50 ° C. or lower, and after taking out the solution, 80 ml of water was added and stirred and mixed. After removing DMF and water with an evaporator, 20 ml of water was added again to obtain a polyacrylic acid-modified titanium dioxide aqueous solution. Titanium dioxide particles were precipitated by adding 1 ml of 2N hydrochloric acid, and unreacted polyacrylic acid was separated by removing the supernatant after centrifugation. Water was added again for washing, and water was removed after centrifugation. After adding 10 ml of 50 mM phosphate buffer (pH 7.0), ultrasonic treatment was performed at 200 Hz for 30 minutes to disperse the titanium dioxide particles. After ultrasonic treatment, the solution was filtered through a 0.45 μm filter to obtain a polyacrylic acid-modified titanium dioxide sol having a solid content of 1.5%. The dispersion particle diameter of the produced polyacrylic acid-modified titanium dioxide fine particles (anatase type) was changed to 0.75 ml of a dispersion containing polyethyleneimine-bound titanium dioxide fine particles in a zeta potential measurement cell using Zeta Sizer Nano ZS (manufactured by Sysmex Corporation). The various parameters of the charged and solvent were set to the same values as in water, and measured by a dynamic light scattering method at 25 ° C. As a result of cumulant analysis, the average dispersed particle size was 45.5 nm.

(Example 2)
Immobilization of anti-AFP antibody molecules on polyacrylic acid modified titanium dioxide fine particles 1 ml of polyacrylic acid modified titanium dioxide sol (anatase type) obtained in Example 1 was subjected to solution exchange using a desalting column PD10 and dispersed in water. 3 ml of polyacrylic acid modified titanium dioxide sol was obtained. To 1.5 ml of this solution, 0.1 ml of a mixture of 200 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 50 mM N-hydroxysuccinimide (NHS) was added and stirred for 10 minutes. The carboxyl group was activated. After stirring, the solution was exchanged using PD10 equilibrated with 10 mM acetate buffer (pH 5.0), and 3 ml of carboxyl group-activated polyacrylic acid modified titanium dioxide sol dispersed in 10 mM acetate buffer (pH 5.0) was obtained. It was. Anti-α-fetoprotein (anti-AFP) polyclonal antibody (goat IgG, SC-8108; Cosmo Bio) prepared in the same buffer was added to a concentration of 0.05 mg / ml. After stirring at room temperature for 15 minutes, an ethanolamine hydrochloride aqueous solution (pH 8.5) was added to a concentration of 0.5M. After stirring for 10 minutes, 1 ml of 2N hydrochloric acid was added to precipitate titanium dioxide particles, and the supernatant was removed after centrifugation. Water was added again for washing, and water was removed after centrifugation. After adding 2.5 ml of 50 mM phosphate buffer (pH 7.0), ultrasonic treatment was performed at 200 Hz for 30 minutes to disperse the titanium dioxide particles. After sonication, the mixture was filtered through a 0.45 μm filter to obtain an anti-AFP antibody-immobilized titanium dioxide composite sol having a solid content of 0.3%. The dispersion particle diameter of the produced anti-AFP antibody-immobilized titanium dioxide complex (anatase type) was measured in the same manner as in Example 1. As a result, it was 52.8 nm. Furthermore, after freeze-drying the dispersion containing the prepared anti-AFP antibody-immobilized titanium dioxide complex overnight, 2.5 ml of 50 mM phosphate buffer (pH 7.0) was added, and ultrasonication was performed at 200 Hz for 30 minutes. The dispersion particle diameter of the treated and dispersed anti-AFP antibody-immobilized titanium dioxide complex was measured in the same manner as in Example 1. As a result, it was 53.7 nm.

(Example 3)
Immobilization of anti-HSA antibody molecules on polyacrylic acid-modified titanium dioxide fine particles 1 ml of polyacrylic acid-modified titanium dioxide sol (anatase type) obtained in Example 1 was subjected to solution exchange using a desalting column PD10 and dispersed in water. 3 ml of polyacrylic acid modified titanium dioxide sol was obtained. To 1.5 ml of this solution, 0.1 ml of a mixture of 200 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 50 mM N-hydroxysuccinimide (NHS) was added and stirred for 10 minutes. The carboxyl group was activated. After stirring, the solution was exchanged using PD10 equilibrated with 10 mM acetate buffer (pH 5.0), and 3 ml of carboxyl group-activated polyacrylic acid modified titanium dioxide sol dispersed in 10 mM acetate buffer (pH 5.0) was obtained. It was. Anti-human serum albumin (anti-HSA) monoclonal antibody (mouse IgG, MSU-304; Cosmo Bio) prepared in the same buffer was added to a concentration of 0.05 mg / ml. After stirring at room temperature for 15 minutes, an ethanolamine hydrochloride aqueous solution (pH 8.5) was added to a concentration of 0.5M. After stirring for 10 minutes, an equal amount of 2.5 M NaCl, 20% (w / v) polyethylene glycol was added to precipitate titanium dioxide particles, and the supernatant was removed after centrifugation. Water was added again for washing, and water was removed after centrifugation. 2.5 ml of PBS buffer (pH 7.0: Nippon Gene containing 100 mM NaCl) was added to disperse the titanium dioxide particles. The mixture was filtered with a 0.45 μm filter to obtain an anti-HSA antibody-immobilized titanium dioxide composite sol having a solid content of 0.3%. The dispersion particle diameter of the produced anti-HSA antibody-immobilized titanium dioxide complex (anatase type) was measured in the same manner as in Example 1. As a result, it was 52.8 nm.

Example 4
Immobilization of Streptavidin Molecules in Polyacrylic Acid-modified Titanium Dioxide Fine Particles 1 ml of polyacrylic acid-modified titanium dioxide sol (anatase type) obtained in Example 1 was subjected to solution exchange using a desalting column PD10 and dispersed in water. 3 ml of acrylic acid modified titanium dioxide sol was obtained. To 1.5 ml of this solution, 0.1 ml of a mixture of 200 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 50 mM N-hydroxysuccinimide (NHS) was added and stirred for 10 minutes. The carboxyl group was activated. After stirring, the solution was exchanged using PD10 equilibrated with 10 mM acetate buffer (pH 5.0), and 3 ml of carboxyl group-activated polyacrylic acid modified titanium dioxide sol dispersed in 10 mM acetate buffer (pH 5.0) was obtained. It was. Streptavidin (Pierce Biotechnology Inc. code: 21126) was added to a concentration of 0.05 mg / ml. After stirring at room temperature for 15 minutes, an ethanolamine hydrochloride aqueous solution (pH 8.5) was added to a concentration of 0.5M. After stirring for 10 minutes, an equal amount of 2.5 M NaCl, 20% (w / v) polyethylene glycol was added to precipitate titanium dioxide particles, and the supernatant was removed after centrifugation. Water was added again for washing, and water was removed after centrifugation. 2.5 ml of PBS buffer (pH 7.0, Nippon Gene) was added to disperse the titanium dioxide particles. The mixture was filtered through a 0.45 μm filter to obtain a streptavidin-immobilized titanium dioxide composite sol having a solid content of 0.3%. When the dispersed particle diameter of the produced streptavidin-immobilized titanium dioxide complex (anatase type) was measured in the same manner as in Example 1, it was 50.5 nm.

Evaluation of pH stability of titanium dioxide composite microparticles Buffers with different pH of 50 mM (pH 4 and 5 = acetic acid buffer, pH 6 = 2-morpholinoethanesulfonic acid buffer), pH 7 and 8 = 2- [4- ( 2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid buffer, pH 9 = borate buffer, pH 10 = glycine sodium hydroxide buffer) to a final concentration of 0.025 (w / v)% The dispersion containing the streptavidin-immobilized titanium dioxide composite fine particles obtained in Example 4 was added to the mixture, and allowed to stand at room temperature for 1 hour. 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 FIG. Although a change in particle size was observed between pH 4 and 10, it was about 75 to 50 nm. Moreover, after leaving these still at room temperature for 24 hours, when the average dispersion | distribution particle size was measured similarly, the change of a particle size was less than 5 nm. It became clear that these showed the stable dispersibility.

Evaluation of salt strength stability of titanium dioxide composite fine particles A dispersion containing the streptavidin-immobilized titanium dioxide composite fine particles obtained in Example 4 in a 10 mM phosphate buffer containing 0.01 to 0.5 M of different sodium chloride was used. The final concentration was 0.025 (w / v)%, and the mixture was allowed to stand at room temperature for 1 hour. Thereafter, the dispersed particle size was measured in the same manner as in Example 1. As shown in FIG. When the salt concentration in the system was 0.01 to 0.5 M, almost no change in the dispersed particle diameter was observed. Moreover, after leaving these still at room temperature for 24 hours, when the average dispersion | distribution particle size was measured similarly, the change of a particle size was less than 5 nm. It became clear that these showed the stable dispersibility.

Evaluation of Uniformity (Transparency) of Titanium Dioxide Composite Fine Particles A dispersion containing the streptavidin-immobilized titanium dioxide composite fine particles obtained in Example 4 was finished using a 10 mM phosphate buffer containing 0.1 M sodium chloride. The concentration was adjusted to 0.1% and the mixture was allowed to stand at room temperature for 1 hour. Further, P25 (Nippon Aerosil) as titanium dioxide fine particles was similarly adjusted to a final concentration of 0.1% using a 10 mM phosphate buffer containing 0.1 M sodium chloride, and allowed to stand at room temperature for 1 hour. I put it. Thereafter, 5 ml was transferred to a petri dish and photographed from above to confirm. The result is shown in FIG. It was confirmed that the dispersion liquid containing streptavidin-immobilized titanium dioxide composite fine particles was clearly highly transparent and uniformly dispersed in the P25 aqueous solution. Further, as a result of measuring the absorbance at a wavelength of 660 nm using a spectrophotometer (UV-1600, Shimadzu Corporation), the absorbance of P25 aqueous solution was much higher than 1, whereas it was impossible to measure streptavidin. The dispersion containing the titanium dioxide composite fine particles had an absorbance of 0.044, and no precipitate was formed. Furthermore, after these solutions were allowed to stand at room temperature in a dark place for 2 weeks, the absorbance at a wavelength of 660 nm was measured in the same manner. As a result, the absorbance of the P25 aqueous solution greatly exceeded 1 and was not measurable. The absorbance of the dispersion containing the streptavidin-immobilized titanium dioxide composite fine particles was 0.051. From this, it became clear that the titanium dioxide composite fine particles exhibit high transparency, uniform dispersibility, and are stable in an aqueous solution.

(Example 5)
Synthesis of polyacrylic acid-modified magnetic composite titanium dioxide fine particles
In a separable flask, 45.16 g of polyoxyethylene (15) cetyl ether (C-15; Nippon Surfactant Kogyo Co., Ltd.) was dissolved and purged with nitrogen for 5 minutes. Then, 75 ml of cyclohexene solution (Wako Pure Chemical Industries) was added. 3.6 ml of a .67M FeCl ₂ (Wako Pure Chemical) aqueous solution was added, and 5.4 ml of a 30% aqueous ammonia solution was added while stirring at 250 rpm, and allowed to react for 1 hour. Thereafter, 0.4 ml of 50 mM tetraethylorthosilicate aqueous solution (Wako Pure Chemical Industries, Ltd.) was dropped and reacted for 1 hour. Thereafter, titanium tetraisopropoxide (Wako Pure Chemical Industries) was added to a final concentration of 0.005M. 10 ml of 50% (w / v) aqueous ethanol solution was added in 1 ml increments at 10 minute intervals. The reaction solution was centrifuged, and the precipitate was calcined at 350 ° C. for 2 hours. After baking, it was dispersed in a 10 mM nitric acid aqueous solution, subjected to ultrasonic treatment, and then filtered through a 0.1 μm filter. The resulting magnetic material: 0.75 ml of composite titanium dioxide sol was dispersed in 20 ml of dimethylformamide (DMF), and 10 ml of DMF in which 0.3 g of polyacrylic acid (average molecular weight: 5000, Wako Pure Chemical Industries) was dissolved was added and stirred. And mixed. The solution was transferred to a hydrothermal reaction vessel (HU-50, Sanai Kagaku) and synthesized at 180 ° C. for 6 hours. After completion of the reaction, the reaction vessel was cooled to a temperature of 50 ° C. or lower, and the solution was taken out into a separatory funnel. Next, 40 ml of chloroform was added and mixed by stirring to remove the lower layer, and the upper layer was recovered. This step was repeated twice to remove DMF. To 10 ml of this solution, 10 ml of 1.5 M NaCl, 20% (w / v) polyethylene glycol 6000 (Wako Pure Chemical Industries) was added, and the supernatant was removed after centrifugation. 2.5 ml of water was added to the precipitate, and gel filtration was performed with a Sephadex G-25 column to obtain a dispersion of polyacrylic acid-modified magnetic material composite titanium dioxide fine particles (anatase type).

(Example 6)
Immobilization of anti-HSA antibody molecules on polyacrylic acid-modified magnetic material composite titanium dioxide fine particles 200 mM 1-ethyl-3 was added to 1.5 ml of the dispersion of the polyacrylic acid-modified magnetic material composite titanium dioxide fine particles obtained in Example 5. 0.1 ml of a mixed solution of-(3-dimethylaminopropyl) carbodiimide and 50 mM N-hydroxysuccinimide (NHS) was added and stirred for 10 minutes to activate the carboxyl group. After completion of stirring, the solution was exchanged using PD10 equilibrated with 10 mM acetate buffer (pH 5.0), and the carboxyl group-activated polyacrylic acid-modified magnetic material composite titanium dioxide sol dispersed in 10 mM acetate buffer (pH 5.0) 3 ml was obtained. An anti-human serum albumin (anti-HSA) monoclonal antibody (mouse IgG: MSU-304, Cosmo Bio) prepared in the same buffer was added to a concentration of 0.05 mg / ml. After stirring at room temperature for 15 minutes, an ethanolamine hydrochloride aqueous solution (pH 8.5) was added to a concentration of 0.5M. After stirring for 10 minutes, an equal amount of 2.5 M NaCl, 20% (w / v) polyethylene glycol was added to precipitate the magnetic material composite titanium dioxide particles, and the supernatant was removed after centrifugation. Water was added again for washing, and water was removed after centrifugation. 2.5 ml of PBS (Nippon Gene) was added to disperse the magnetic material composite titanium dioxide particles. The mixture was filtered through a 0.45 μm filter to obtain a magnetic material composite titanium dioxide composite sol having an anti-HSA antibody-immobilized solid content of 0.3%. When the dispersed particle diameter of the produced anti-HSA antibody-immobilized magnetic material composite titanium dioxide composite (anatase type) was measured in the same manner as in Example 1, it was 105 nm.

(Example 7)
Introduction of acrylic acid / sulfonic acid copolymer into titanium dioxide particles 0.75 ml of a 20% solid component titanium dioxide sol (anatase type) obtained in Example 1 was dispersed in 20 ml of dimethylformamide (DMF) and acrylic. After adding 10 ml of DMF in which 0.3 g of an acid / sulfonic acid monomer copolymer (average molecular weight: 5000, sample freeze-dried after proton substitution, Nippon Shokubai) was added, the mixture was stirred and mixed. The solution was transferred to a hydrothermal reaction vessel (HU-50, Sanai Kagaku) and reacted at 150 ° C. for 5 hours. After completion of the reaction, the reaction vessel was cooled to room temperature, and twice the amount of isopropanol (Wako Pure Chemical Industries) was added to the reaction solution. After leaving still at room temperature for 30 minutes or more, centrifugation was performed at 4000 × g for 20 minutes to collect a precipitate. This precipitate was washed with 70% ethanol, and then 2.5 ml of water was added to obtain an acrylic acid / sulfonic acid copolymer-modified titanium dioxide sol (anatase type).

(Example 8)
Immobilization of anti-DR4 antibody molecules on acrylic acid / sulfonic acid copolymer-modified titanium dioxide fine particles 200 ml of 1-ethyl-200 mM was added to 1.5 ml of acrylic acid / sulfonic acid copolymer-modified titanium dioxide sol prepared in Example 7. 0.1 ml of a mixed solution of 3- (3-dimethylaminopropyl) carbodiimide and 50 mM N-hydroxysuccinimide (NHS) was added and stirred for 10 minutes to activate the carboxyl group. After completion of stirring, the solution was exchanged using PD10 equilibrated with 10 mM acetate buffer (pH 5.0), and the carboxyl group-activated acrylic acid / sulfonic acid copolymer dispersed in 10 mM acetate buffer (pH 5.0) 3 ml of modified titanium dioxide sol was obtained. To this, an anti-DR4 monoclonal antibody (Anti-TRAIL Receptor 1, mouse, code: SA-225, Funakoshi) was added to a concentration of 0.05 mg / ml. After stirring for 1 minute at room temperature, an ethanolamine hydrochloride aqueous solution (pH 8.5) was added to 0.5 M. After stirring at room temperature for 10 minutes, an equal amount of 2.5 M NaCl and 20% (w / v) polyethylene glycol was added to precipitate titanium dioxide particles, and the supernatant was removed by centrifugation. After washing with water and centrifugation, the precipitate was collected, and 2.5 ml of PBS buffer (pH 7.0, Nippon Gene) was added to disperse the titanium dioxide particles. Filtration through a 0.45 μm filter gave an anti-DR4 antibody-immobilized titanium dioxide composite sol (anatase type) having a solid content of 0.3%.

Example 9
Degradation of antigen AFP by anti-AFP antibody-immobilized titanium dioxide complex α-fetoprotein (AFP, Cosmo Bio) was diluted with 50 mM PBS buffer (pH 7.0, Nippon Gene) to 1 μg / ml. The anti-AFP antibody-immobilized titanium dioxide composite prepared in Example 2 was added so that the solid component was 0.01%. Next, the mixture was allowed to stand at 37 ° C. for 3 hours to form an aggregate by an antigen-antibody reaction. Since AFP and the anti-AFP antibody-immobilized titanium dioxide complex formed an aggregate, it is clear that the anti-AFP antibody-immobilized titanium dioxide complex specifically recognizes and binds to AFP. The agglomerate was stirred and irradiated with ultraviolet light having a wavelength of 340 nm so as to have a wavelength of 1 mW / cm ² , and absorption (wave turbidity of the aggregate) at 600 nm was measured with a spectrophotometer. The results are shown in FIG. Only at the time of ultraviolet (UV) irradiation, a decrease in absorbance with a decrease in aggregate concentration is observed. That is, it was revealed that the antigen AFP is decomposed by the photocatalytic action of the anti-AFP antibody-immobilized titanium dioxide complex.

(Example 10)
Confirmation of antigen-antibody reaction by anti-HSA antibody-immobilized titanium dioxide complex Human serum albumin (HSA, Cosmo Bio) was diluted with 50 mM PBS buffer (pH 7.0, Nippon Gene) to 250 μg / ml. . Separately, a surface plasmon resonance sensor chip C1 (BIACORE) was activated with a mixture of 400 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 100 mM N-hydroxysuccinimide (NHS). . This sensor chip is attached to a surface plasmon resonance measuring apparatus: BIACORE1000 (BIACORE), the previous HSA solution is passed at a flow rate of 10 μl / min, and active groups are blocked with 0.1 M ethanolamine to immobilize HSA. A sensor chip was produced. 0.01% anti-HSA antibody-immobilized titanium dioxide composite sol produced in Example 3 and 0.01% streptavidin-immobilized titanium dioxide composite sol produced in Example 4 to this HSA-immobilized sensor chip The antigen-antibody reaction was confirmed. The results are shown in FIG. The anti-HSA antibody-immobilized titanium dioxide complex reacted and bound to the chip against the HSA-immobilized sensor chip, but the streptavidin-immobilized titanium dioxide complex did not react and binding did not occur. That is, it was confirmed that the anti-HSA monoclonal antibody immobilized on the hydrophilic polymer on titanium dioxide surely retained the activity as an antibody even after immobilization.

(Example 11)
Degradation of antigen HSA by anti-HSA antibody-immobilized titanium dioxide complex Anti-HSA antibody-immobilized dioxide prepared in Example 3 by diluting HSA to 20 ng / ml with PBS buffer (pH 7.0, Nippon Gene) The titanium composite was added so as to have a solid content of 0.01%. Next, after standing at room temperature for 30 minutes, ultraviolet light with a wavelength of 340 nm was irradiated so as to be 1 mW / cm ² , and sampling was performed every 15 minutes for 90 minutes. The same treatment was performed on the streptavidin-immobilized titanium dioxide composite produced in Example 4. Separately, an anti-HSA polyclonal antibody (rabbit) immobilized sensor chip for surface plasmon resonance measurement was prepared according to the method of Example 8. Using BIACORE1000 in the same manner as in Example 8, 20 μl of each time-lapse sample was fed to an anti-HSA antibody-immobilized sensor chip, and then 10 μl of 50 μg / ml of anti-HSA polyclonal antibody (rabbit) was fed as a secondary antibody to sandwich assay. Then, the RU value (corresponding to the binding amount) 10 seconds after the antibody feeding was measured. FIG. 4 shows the decomposition rate of HSA calculated from the relative value when the RU value when UV is not irradiated is 100%. From the results of FIG. 4, it was shown that the anti-HSA antibody-immobilized titanium dioxide complex has a very high degradation rate of HSA compared to the streptavidin-immobilized titanium dioxide complex.

(Example 12)
Preparation of liposome encapsulating titanium dioxide composite fine particles Dipalmitoyl phosphatidylcholine (DPPC) / Cholesterol (Chol) / Dipalmitoyl phosphatidylic acid (DPPA) / Maleimidated dipalmitoyl phosphatidylethanolamine (maleimidated DPPE) = 18 / Add 1 ml of 0.4M citrate buffer pH 4 to 100 mg of a solid mixture (Nippon Seika) consisting of 10/2 / 0.5 (mol ratio) and stir. Lamella liposomes were prepared. A dispersion containing the streptavidin-immobilized titanium dioxide composite fine particles obtained in Example 4 was obtained by evaporating and distilling 10 ml of the solution containing the aforementioned multilamellar liposome dissolved in chloroform by a rotary evaporator and drying the formed lipid film under reduced pressure. The solution was added, vortexed, and further sonicated to prepare liposomes encapsulating titanium dioxide composite particles. Next, the obtained titanium dioxide composite microparticle-encapsulated liposome was heated 10 times with a pressure device (extruder, biomembranes) equipped with a polycarbonate membrane (Nucleopore, Microscience) having a pore size of 200 nm while being heated 10 times. Repeated pressure filtration yielded sized liposomes. Thereafter, the average dispersed particle size was measured in the same manner as in Example 1. As a result, it was confirmed that the dispersed particle size was 185.7 nm.

  The present invention relates to endocrine disrupting substances, or biomolecules such as proteins, antibodies and DNA having molecular recognition ability against pathogenic molecules and cancer cells, by immobilizing them on titanium dioxide modified with a water-soluble polymer, and thereby molecules for these. Dispersion liquid containing a titanium dioxide complex having a recognition ability and a molecular discrimination ability showing a decomposition reaction of these substances by photocatalytic action such as ultraviolet irradiation. The titanium dioxide complex in this dispersion has the ability to specifically recognize and capture the target substance and to strongly decompose the target substance by ultraviolet irradiation or the like. This is useful for, for example, decomposing endocrine disrupting substances in aqueous systems. In addition, when used in a living body, the ability to maintain a highly dispersed state under physiological conditions, the ability to accurately capture a target substance, and the ability to have a strong photocatalytic activity make it an application to DDS and further selective cancer. It is extremely useful for medical applications such as cell destruction.

FIG. 1 is a schematic view showing a titanium dioxide complex having molecular discrimination ability of the present invention. The titanium dioxide fine particles (1) are modified with the hydrophilic polymer (2), and the molecules (3) having specific binding ability are immobilized on the hydrophilic polymer (2). FIG. 2 is a view showing the antigen (α-fetoprotein) degrading activity of the anti-α-fetoprotein antibody-immobilized titanium dioxide complex of the present invention. Antigen degradation activity is expressed as a decrease in absorbance. FIG. 3 is a diagram showing the results of evaluating the binding properties of the anti-human serum albumin antibody-immobilized titanium dioxide complex of the present invention and an antigen (human serum albumin) by the surface plasmon resonance method. As a control, streptavidin-immobilized titanium dioxide complex was used. FIG. 4 is a view showing the results of the activity of decomposing antigen (human serum albumin) by the anti-human serum albumin antibody-immobilized titanium dioxide complex of the present invention. The degradation activity of the antigen is displayed as a degradation rate (%) calculated from a decrease in the amount of binding of the antigen to the antibody accompanying the degradation (measured by the surface plasmon resonance method). As a control, streptavidin-immobilized titanium dioxide complex was used. FIG. 5 is a diagram showing the dispersibility of the solution with respect to pH change by the streptavidin-immobilized titanium dioxide composite of the present invention. The average dispersed particle size of the titanium dioxide composite is plotted against pH change. FIG. 6 is a diagram showing the dispersibility of the streptavidin-immobilized titanium dioxide composite of the present invention with respect to the salt concentration change of the solution. The average dispersed particle size of the titanium dioxide composite is plotted against the salt concentration change. FIG. 7 is an image taken from above of the state of a dispersion containing the streptavidin-immobilized titanium dioxide composite of the present invention and a solution containing untreated titanium dioxide fine particles (P25).

Claims (18)

The surface of the titanium dioxide fine particles is modified with a hydrophilic polymer having a carboxyl group, the carboxyl group of the hydrophilic polymer and titanium dioxide are bonded by an ester bond, and the carboxyl residue of the hydrophilic polymer is bonded to the carboxyl residue. A titanium dioxide composite comprising a titanium dioxide composite and a dispersion medium, in which a molecule having a specific binding ability to a target molecule is immobilized, wherein the titanium dioxide fine particles have a particle size of 2 to 200 nm Dispersion. The dispersion liquid according to claim 1, wherein the dispersion medium is an aqueous solution that is allowed to be introduced into a living body, and the titanium dioxide composite is contained in the dispersion medium. The dispersion liquid according to claim 2, wherein the dispersion medium has a pH of 4 to 10. The dispersion liquid according to claim 3, wherein the dispersion medium is a pH buffer solution. The dispersion liquid according to any one of claims 3 to 4, wherein the dispersion medium has a salt concentration of 1 M or less. The dispersion liquid according to claim 5, wherein the dispersion medium is physiological saline. The dispersion according to any one of claims 1 to 6, wherein the titanium dioxide composite fine particles are included in a package that is allowed to be introduced into a living body. The dispersion according to claim 7, wherein the inclusion body is any one of a liposome, a virus particle, and a hollow nanoparticle. The dispersion according to any one of claims 1 to 8, wherein the titanium dioxide is anatase type or rutile type. The dispersion according to any one of claims 1 to 9, wherein the titanium dioxide is composite titanium dioxide composed of titanium dioxide and a magnetic material. The dispersion liquid according to claim 1, wherein the hydrophilic polymer is a water-soluble polymer. The dispersion according to claim 11, wherein the water-soluble polymer contains a polycarboxylic acid. The dispersion according to claim 11, wherein the water-soluble polymer contains a copolymer having a plurality of carboxyl group units in the molecule. The dispersion according to any one of claims 1 to 13, wherein the molecule having a specific binding ability to the target molecule is an amino acid, a peptide, a simple protein, a complex protein, or an antibody. liquid. The dispersion liquid according to any one of claims 1 to 13, wherein the molecule having a specific binding ability to the target molecule is a nucleoside, a nucleotide, a nucleic acid, and a peptide nucleic acid. The dispersion according to any one of claims 1 to 13, wherein the molecule having a specific binding ability to the target molecule is a monosaccharide, a sugar chain, a polysaccharide, and a complex carbohydrate. liquid. The dispersion liquid according to any one of claims 1 to 13, wherein the molecule having a specific binding ability to the target molecule is a fatty acid, a fatty acid derivative, a simple lipid, or a complex lipid. A pharmaceutical comprising the dispersion according to any one of claims 1 to 17.

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