JP2008195653A - Composite particle used for drug delivery system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composite particle producing hydroxy radicals in an aqueous solution by irradiation of ultrasonic waves and further making dispersion stability under neutral physiological conditions in a living body compatible with functions to specifically recognize a specified biological tissue in a well-balanced state. <P>SOLUTION: The composite particle has TiO<SB>2</SB>particles producing the hydroxy radicals in the aqueous solution by irradiation of the ultrasonic waves, a polyacrylic film coating at least a part of the surface of the TiO<SB>2</SB>particles and a polypeptide containing a preS1/S2 sequence of a hepatitis B virus bound to the polyacrylic film and specifically recognizing a liver tissue of a mammal. The dispersion particle diameter is within the range of ≥50 to ≤200 nm. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a composite particle used in a drug delivery system.

In recent years, attention has been focused on application development in various fields using TiO ₂ as a photocatalyst. It utilizes the oxidizing power of reactive oxygen species generated by irradiating TiO ₂ with light with a short wavelength of 380 nm or less, and has been reported to decompose harmful chemical substances such as environmental hormones, and to kill and antibacterial harmful microorganisms. ing. Furthermore, research as a novel cancer treatment is advanced using this photocatalytic reaction (see Non-Patent Documents 1 to 4).

In the laboratory to which the present inventors belong, it has been confirmed that when TiO ₂ , which is known as such a photocatalyst, is irradiated with ultrasonic waves, a high concentration of hydroxyl radical (.OH) is produced (titanium dioxide / super Sonic catalysis method (TiO _{2 /} US method)). TiO ₂ / U. S. The method has advantages such as a longer ultrasonic penetration distance into the aqueous phase than the photocatalytic reaction by utilizing ultrasonic waves, and can be expected to be applied in various fields. To date, this TiO ₂ / U. S. It was confirmed that effective bactericidal effects such as Escherichia coli and Legionella can be obtained by using the method.

  In addition, for cancer treatment using ultrasonic waves, those using a heating action by absorption of ultrasonic waves in living tissue, those using a mechanical action by ultrasonic vibration, and the cavitation effect caused by ultrasonic waves It has been reported that there are sonochemotherapy that causes a chemical reaction to a substance administered to the substance, and that when cancer cells are irradiated with ultrasonic waves, apoptosis is induced to suppress the proliferation of the cancer cells.

R, Cai, Y. Kubota, T. Shuin, et al. Induction of Cytotoxicity by Photoexcited TiO2 Particles. Cancer Res. 52 (1992) 2346-2348. Photocatalytic killing effect of TiO2 nanoparticles on Ls-174-t human.colon carcinoma cells, World J Gastroenterol 10 (2004) 3191-3193. Q. Liu, X. Wang, P. Wang, et al. Sonodynamic effrct of protoporphyrin IX disodium salt on isolated sarcoma 180 cells.Ultrasonics 2006; 45: 56-60. H.Honda, Q.L.Zhao, T.Kondo.Effects Of Dissolved Gases And Echo Contrast Agent On Apotosis Induced By Ultrasound And Its Mechanism Via The Mitochondria-Caspaze Pathway.Ultrasound in Med. & Biol.

However, the prior art described in the above literature has room for improvement in the following points.
First, TiO ₂ particles used in the above-described titanium dioxide / ultrasonic catalyst method have insufficient dispersion stability under neutral physiological conditions in the living body. It was difficult to ensure fluidity.

Secondly, TiO ₂ particles used in the above-mentioned titanium dioxide / ultrasonic catalyst method are difficult to specifically recognize a specific living tissue. It was difficult to generate high concentration of hydroxyl radicals (.OH) intensively in living tissue.

  The present invention has been made in view of the above circumstances, and in addition to being able to generate hydroxy radicals in an aqueous solution by ultrasonic irradiation, further, dispersion stability and identification under neutral physiological conditions in vivo. An object of the present invention is to provide composite particles that can balance both functions of specifically recognizing a living tissue in a well-balanced manner.

  According to the present invention, the substrate particles that generate hydroxy radicals in an aqueous solution by ultrasonic irradiation, the polymer film that covers at least a part of the surface of the substrate particles, and the polymer film are bonded to A composite particle comprising a polypeptide that specifically recognizes a biological tissue is provided.

  Since the composite particle has a polymer film that covers at least a part of the surface of the base particle, a polypeptide that specifically recognizes a specific biological tissue can be bound to the base particle. As a result, the composite particles can be specifically transferred to a specific biological tissue in vivo, and can generate high-concentration hydroxy radicals in the specific biological tissue by irradiation with ultrasonic waves from outside the living body. It becomes possible. Therefore, if this composite particle is used, cell damage of a specific living tissue can be promoted by irradiation with ultrasonic waves from outside the living body.

  According to the composite particle of the present invention, cell damage of a specific living tissue can be promoted by irradiation with ultrasonic waves from outside the living body.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings, the same reference numerals are given to the same components, and the description will be omitted as appropriate.

<Overview>
The composite particles according to the present embodiment are bonded to a base particle that generates hydroxy radicals in an aqueous solution by ultrasonic irradiation, a polymer film that covers at least part of the surface of the base particle, and the polymer film. And a composite particle comprising a polypeptide that specifically recognizes a specific living tissue.

  As described above, since the composite particle according to the present embodiment has a polymer film that covers at least a part of the surface of the base particle, a polypeptide that specifically recognizes a specific biological tissue is bound to the base particle. be able to. As a result, the composite particles can be specifically transferred to a specific biological tissue in vivo, and can generate high-concentration hydroxy radicals in the specific biological tissue by irradiation with ultrasonic waves from outside the living body. It becomes possible. Therefore, if this composite particle is used, cell damage of a specific living tissue can be promoted by irradiation with ultrasonic waves from outside the living body.

<Substrate particles>
As the substrate particles in the present embodiment, any particles can be suitably used as long as they can generate active oxygen, particularly hydroxy radicals (.OH) in an aqueous solution by ultrasonic irradiation. Oxide semiconductor catalyst particles can be used. As the oxide semiconductor catalyst particles, granular ZnO, TiO ₂ , SnO ₂ , Ta ₂ O ₃ , NiO, FeO, CrO, Cr ₂ O ₃ , MoO _{3 and the} like can be used. In particular, rutile-type TiO ₂ catalyst particles or anatase-type TiO ₂ catalyst particles are preferable because they are excellent in hydroxyl radical generating ability.

  The average dispersed particle size of the substrate particles in this embodiment is preferably 50 nm or more and 200 nm or less under physiological conditions. If the average dispersed particle size of the substrate particles is within this range, the complex particles obtained by the production method described later are likely to be within the same range, and therefore a polypeptide that specifically recognizes a specific living tissue. This is because it is large enough to bind to each other via a polymer coating, and sufficient dispersion stability and fluidity can be secured in an aqueous solution such as in the bloodstream of mammals.

Here, the average dispersed particle diameter means an average value calculated by a cumulant method analysis after measurement by a dynamic light scattering method. The physiological condition referred to here is a phosphate buffered saline having a composition of (137 mM NaCl, 8.1 mM Na ₂ HPO ₄ , 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ ) at 25 ° C. and 1 atm. This indicates the presence of water (pH 7.4).

<Titanium Dioxide / ultrasonic irradiation method TiO ₂ / U. S. Law>
Photocatalytic photocatalysts are attracting attention in many industrial fields, and application development for practical use is actively performed.

  First, a catalyst is a substance that does not change itself during a chemical reaction and stops the reaction. By using a catalyst, the activation energy can be reduced, and the reaction can proceed with less energy. Further, since the catalyst itself does not change, in principle, it can be used semipermanently.

A photocatalyst is a catalyst that starts to work when irradiated with light and that works by the energy of light. A photocatalyst absorbs light and enters a high energy state. The energy is given to the reactant to cause a chemical reaction. ).
-Reactions that are unlikely to occur at temperatures as high as tens of thousands of degrees can occur at room temperature.
・ In a normal chemical reaction, when the reaction starts, the reaction proceeds until the reactants disappear. On the other hand, if the light irradiation is stopped, the reaction can be stopped.
-It will not work without light, and will not react unless the reactants come into contact with the photocatalyst.

Definition of Photocatalyst A photocatalyst, which is a kind of semiconductor, is defined as a substance that does not change before and after the reaction but promotes the reaction by absorbing ultraviolet light. If the reaction by the photocatalyst (photocatalytic reaction) 1972, the Honda-Fujishima et al. Irradiating light to TiO ₂ in an aqueous solution, water is found by decomposing into oxygen and hydrogen, it is called Honda Fujishima effect Yes. Attention is now being paid to the use of this photocatalytic reaction for the degradation of environmental pollutants such as dioxins, antibacterial and bactericidal effects, and deodorization.

Titanium dioxide (TiO ₂ )
TiO ₂ is a very stable substance that does not dissolve in acids, alkalis, and organic solvents at normal temperature and pressure and does not react with highly reactive gases such as hydrogen fluoride, chlorine, and hydrogen sulfide. In addition, it has many advantages in terms of durability, wear resistance, economy, safety, practicality, etc., so it is mainly used as a photocatalyst and research is being conducted.

TiO ₂ has three types of crystal structures, a rutile type, an anatase type, and a brookite type (rutile type and anatase type crystal structures are shown in FIG. 2). Of these, the rutile type is the most stable crystal form, and the anatase type and brookite type change to the rutile type at high temperatures. TiO ₂ is a perfect insulator at room temperature, but becomes a semiconductor when irradiated with light or heated. The rutile-type band gap is 3.0 eV, the anatase-type and brookite-type band gaps are 3.2 eV, and the energy is equivalent to 390 nm near-ultraviolet light when converted to the wavelength of light. A highly active anatase type is used as a photocatalyst, and a rutile type having a low activity is widely used as a white pigment or paint. The other brookite type is a naturally occurring ore that has recently been industrially synthesized, but there have been few studies so far and it is not well understood.

<Photocatalytic reaction>
The photocatalytic mechanism of the reaction mechanism TiO ₂ is shown in FIG.

When TiO2 is irradiated with light in the near-ultraviolet region of 390 nm or less, electrons (e ⁻ ) in the valence band are excited and moved to the conduction band. Holes (h ⁺ ) are generated where the electrons were originally. These electrons and holes have a very strong reducing power and oxidizing power, and react with water to react with water, such as hydroxyl radicals (.OH) and superoxide anions (O ₂ ⁻ ), such as reactive oxygen species. Produce. The reaction formula is shown below.

Among these active oxygen species, the hydroxyl radical has particularly high oxidizing power, and the C—C bond (83 kcal / mol), C—H bond (99 kcal / mol), and C—N bond (73 kcal / mol) in the molecule constituting the organic substance. ), The energy of the hydroxyl radical (120 kcal / mol) is much larger than the binding energy of the C—O bond (84 kcal / mol), the O—H bond (111 kcal / mol), and the N—O bond (93 kcal / mol). These bonds are easily broken. Thus, it is considered that the strong oxidizing power of the active oxygen species generated on the surface of TiO ₂ by irradiating light exerts decomposition / antibacterial action of organic compound substances and harmful gas phase substances which are difficult to decompose.

Reactive oxygen species (ROS)
Active oxygen is oxygen molecules ( ³ O ₂ ) contained in the atmosphere that have been converted to more reactive compounds by obtaining electrical energy or light energy such as ultraviolet rays (by Toshikazu Yoshikawa: All of active oxygen and free radicals (2000) Maruzen and Yoshikawa Kono, see: Free radical science (2004) See Kodansha).

These representative radicals include:
・ Singlet oxygen ( ¹ O ₂ )
・ Superoxide anion radical (O ₂・^- : Common name Superoxide)
・ Hydrogen peroxide (H ₂ O ₂ )
・ Hydroxyl radical (・ OH)
There are four.

Hydroxyl radical (OH)
The hydroxyl radical has a much stronger oxidizing power due to chlorine, ozone, etc., and it can be considered that it reacts with most compounds at a rate close to diffusion rate control with high reactivity. High reactivity means that the hydroxyl radical is unstable. In vivo, it is easy to damage DNA, protein or lipid. However, various harmful chemical substances in water and chemical substances in air can be decomposed and detoxified, and can be used for various applications in environmental fields such as water treatment, deodorization, air purification, antibacterial and antifungal. It is.

  Hydroxyl radicals can be generated by Fenton reaction, ultraviolet irradiation method, ultrasonic irradiation method and the like.

The ultrasonic photocatalytic reaction is characterized in that the reaction proceeds only on the surface of the photocatalyst, and further the reaction proceeds only in the portion irradiated with ultraviolet rays. However, when considering the purification of water and the like, it is difficult to irradiate all the parts with light, which is not necessarily an advantage. The light that can be used by TiO ₂ is limited to ultraviolet rays, and the cost of artificially produced ultraviolet photons is higher than expected. Therefore, in the experiments in Examples described later, decomposition of harmful substances is examined by using ultrasonic waves. Ultrasonic waves can be used in places where light does not reach, such as underground, in vivo, and high-concentration contaminated water, because of its high diffraction power (Fig. 4). In the following, the properties of ultrasound are described.

Characteristics of Ultrasound Sound in the frequency range (approximately 20 Hz to 20 kHz) that can be heard by humans is called audible sound, sound having a frequency lower than 20 Hz is called ultra-low frequency sound, and sound having a frequency exceeding 20 kHz is called ultrasound. Ultrasound has high directivity and can propagate regardless of gas, liquid, or solid. However, when the density of media is extremely different, the sound wave is reflected. These properties are used in information exploration technologies such as seafloor surveying and fish finder. In addition, since the sound wave has a high diffractive power, even if there is an obstacle in the traveling direction, it is possible to irradiate the sound wave behind it by turning around.

  Since ultrasonic waves can generate strong energy sound waves, they are also applied to precision machine cleaning and sterilization.

  Moreover, although electromagnetic waves and light are transverse waves, there are two types of ultrasonic waves: longitudinal waves and transverse waves. When the longitudinal wave propagates in the liquid, a coarse portion and a dense portion are generated in the liquid due to high and low sound pressure. This causes cavitation.

When cavitation effect ultrasonic waves are irradiated, the molecules vibrate in the longitudinal direction due to the influence of longitudinal waves, so that high density and low density states, that is, a high pressure region and a low pressure region can be formed. When solvent molecules are separated from each other in a low pressure state, a cavity is formed where the pressure in the solvent is sufficiently lower than its vapor pressure. At that time, a solvent or solute is formed in the cavity. Of steam is taken in. A relatively small cavity re-dissolves when moving from a low pressure to a high pressure range, but a large cavity allows the solvent to be taken in as a vapor when the bubble surface expands / contracts as the pressure changes in the liquid. A cavity that is sufficiently large to expand and cannot maintain itself, and reaches a critical state, collapses rapidly when it is drawn to the part of the pressure belly. This is shown in FIG. It is said that the pressure and temperature generated locally by this crushing are extremely large, reaching a few hundreds of atmospheres at a pressure and reaching 5000 to tens of thousands of degrees at a temperature. In addition, it has been reported that OH is generated during crushing. This reaction is shown below.

  The ease with which cavitation occurs, that is, the pressure at which cavitation occurs, depends on the physical properties such as the viscosity, vapor pressure, and surface tension of the liquid and the conditions of ultrasonic irradiation. In general, cavitation is more likely to occur at lower frequencies.

  “Ultrasonic cleaning” is the one that most directly reflects this cavitation effect. Water molecules are decomposed in a high-temperature reaction field caused by cavitation that occurs when water is irradiated with ultrasonic waves, and .H and .OH are generated, thereby removing dirt.

A short-lived high-temperature / high-pressure field generated by sonochemistry cavitation is called a hot spot. This unique environment is attracting attention as an extreme reaction field, and a chemical reaction field under the influence of this reaction field is called sonochemistry.

  Since sonochemistry can react locally without contact, it has a high potential for application in the medical and industrial fields. The chemical reaction in sonochemistry is the first step in generating radicals. This radical is generated by irradiating ultrasonic waves into the solution to break the chemical bonds of the molecules in the solution. The reaction is shown below.

In the laboratory to which the present inventors belong, it has been found that high concentration of hydroxyl radical (.OH) is produced by irradiating TiO ₂ with ultrasonic waves.

  The frequency range that can be heard by humans is about 20 Hz to 20 kHz, and a sound having a frequency higher than 20 kHz is called an ultrasonic wave. Ultrasound has high directivity and can propagate regardless of gas, liquid, or solid. In addition, since the sound wave has a high diffraction power, even if there is an obstacle in the traveling direction, it is possible to irradiate the sound wave behind it by turning around. Therefore, it can be used in places where light does not reach, such as underground, highly contaminated water, and living organisms.

<Polypeptide that specifically recognizes specific living tissue>
In the present embodiment, the polypeptide having a specific binding ability that imparts the ability to discriminate a specific biological tissue to the composite particle is a polypeptide that specifically binds to a molecule present in the target tissue. It is not particularly limited. According to this embodiment, functional proteins, structural proteins, antibodies, ligands, receptors, and oligopeptides can be suitably immobilized as polypeptides. In addition, amino groups and thiol groups can be used for immobilizing simple proteins to titanium dioxide complexes, and aldehyde groups of sugars can be used as target functional groups for immobilization in the case of glycoproteins. In addition, for example, 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). It is also possible to fix it by using it.

  In this embodiment, the polypeptide having specific binding ability may include an amino acid sequence derived from a fluorescent protein. This is because it can be confirmed by immunofluorescence or the like whether or not the composite particles are surely delivered to a specific tissue intended for DDS. As the amino acid sequence derived from fluorescent protein, for example, general GFP (green fluorescent protein derived from Aequorea jellyfish), amino acid sequence derived from DsRed, and the like can be preferably used.

  In the present embodiment, the polypeptide having specific binding ability may include an amino acid sequence derived from a tag polypeptide. This is because the polypeptide can be easily purified when producing a polypeptide having a specific binding ability. As an amino acid sequence derived from a tag polypeptide, for example, an amino acid sequence such as GST tag, His tag, TF tag, HA tag, MBP tag, or c-Myc tag, which are general tag polypeptides, is preferably used. Can do.

<Hepatitis B virus>
Drug Delivery System (DDS)
The drug delivery system (DDS) is a system that efficiently delivers a necessary amount of a drug to an affected area. By using DDS, it is possible to sufficiently exert a therapeutic effect and minimize side effects. In recent years, research and development of DDS has been conducted to improve the patient's QOL (Quality of Life) by releasing the side effects associated with the efficacy of the drug, and to commercialize existing drugs that have high therapeutic effects but strong side effects. It has been activated. In particular, targeting (targeting) for controlling the destination in the body by including a drug in an appropriate carrier has become an important research subject (see Non-Patent Document 1).

  In recent years, gene therapy has attracted attention as a new treatment method. In gene therapy, a gene that is deficient in a specific organ or a gene that does not function is replaced with a normal gene. However, if a gene is introduced into other than the target organ, unexpected side effects may occur. Therefore, even in gene therapy, DDS capable of pinpoint targeting is indispensable.

  Currently, hollow protein nanoparticles using viral proteins are being developed as drug and gene carriers. Hollow nanoparticles exhibit the same high gene transfer rate as viruses, and are as safe as liposomes because they are artificial systems. Since it has a higher targeting ability, it is expected to be used as a drug carrier.

Hepatitis B virus envelope Hepatitis B virus has a structure in which the viral genome is encapsulated in nanocapsules made of protein (hepatitis B virus surface antigen protein) and lipid (FIG. 1a). A hollow nanoparticle consisting of protein and lipid without this viral genome is called hepatitis B virus envelope (FIG. 1b).

The method for producing the hepatitis B virus envelope is as follows.
1) The gene for hepatitis B virus surface antigen protein is expressed in yeast cells.
2) Crush the yeast cells and centrifuge the crushed cells.
3) Perform cesium density gradient centrifugation.
4) Perform sucrose density gradient centrifugation.

  In this way, high purity particles can be purified. The yield is about 20 mg of particles per liter of yeast culture. Thus, it has been clarified that the hepatitis B virus envelope particles produced in yeast have no danger to the human body.

  The protein forming the hepatitis B virus envelope consists of three sites: pre-S1 (108 or 119 amino acid residues), pre-S2 (55 amino acid residues), and S protein (226 amino acid residues). Hepatitis B virus envelope particles are uneven in size and have a particle size of about 50-500 nm. The average particle has a particle diameter of 80 nm, and it is clear that it is composed of about 110 hepatitis B virus surface antigen proteins.

  The hepatitis B virus envelope is a hollow nanoparticle that retains the high infectivity of the virus inherent in the liver, and can be specifically introduced into the liver if a gene or drug is included. In addition, by replacing the molecular recognition site of the hepatitis B virus envelope with a molecule that specifically recognizes another organ, it becomes possible to deliver a gene or drug to any organ (retargeting). Currently, the development of retargeting technology is progressing, and it is expected that pinpoint genes and drugs can be freely introduced into any organ.

<Polymer coating>
In the present embodiment, at least a part of the surface of the base particle is coated with the polymer film. For this reason, it becomes possible to bind a polypeptide that specifically recognizes a specific living tissue via a polymer coating to the surface of a base particle, which is normally difficult to directly bind the polypeptide. .

The polymer film used in the present embodiment is preferably a hydrophilic cationic polymer. As described above, the hydrophilic cationic polymer is firmly bonded to the surface of the base particle, thereby having a positive charge near neutrality. Therefore, extremely good dispersion stability is exhibited not only in the vicinity of neutrality but also in an aqueous solvent having a wide pH range. In this case, when the base particles are TiO ₂ particles, the dispersibility becomes extremely good in various pH buffer solutions containing water or salt under physiological conditions without addition of other substances such as a dispersant. Stable dispersion is achieved over time.

  As a material for the polymer film used in this embodiment, a water-soluble polymer having a carboxyl group is desirable. As the water-soluble polymer, any amine having a weight average molecular weight in the range of 1,000 to 100,000 can be used. In addition, any polymer having a carboxyl group can be used, and examples thereof include carboxymethyl starch, carboxymethyl dextran, carboxymethyl cellulose, polycarboxylic acids, and a copolymer (copolymer) having a carboxyl group unit. It is done. Specifically, from the viewpoint of hydrolyzability and solubility of the water-soluble polymer, 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 a hydrophilic polymer having these carboxyl groups, it is possible to immobilize a polypeptide that specifically recognizes a desired specific biological tissue at the carboxyl residue of the hydrophilic polymer. Become. Furthermore, even after immobilization of a polypeptide that specifically recognizes a specific biological tissue, the composite particles were uniformly dispersed in a wide pH range including near neutrality due to the electric repulsion between the remaining carboxyl groups. The state can be maintained.

  As a material for the polymer film used in this embodiment, a water-soluble polymer that is an amine is also desirable. As the water-soluble polymer, any amine having a weight average molecular weight in the range of 1,000 to 100,000 can be used. For example, polyamino acids, polypeptides, polyamines, and copolymers having amine units (copolymers). ) And the like. Specifically, polyamines such as polyethyleneimine, polyvinylamine, and polyallylamine are more preferably used from the viewpoint of hydrolyzability and solubility of the water-soluble polymer.

  Also in this case, the dispersion liquid of this embodiment improves the dispersibility in an aqueous solvent. This is because, in an aqueous dispersion medium, for example, an amine present on the surface of the photocatalytic titanium dioxide fine particles is stably present over a long period without agglomeration due to an electric repulsion between the particles. Moreover, it is basically very stable against pH fluctuations and addition of inorganic salts. Further, the isoelectric point of the photocatalytic titanium dioxide fine particles of the present invention reflects the isoelectric point of the amine of the hydrophilic polymer, and works between the particles as the pH decreases in an aqueous dispersion medium having a pH of 9 or less. Since the electric repulsion increases, it exhibits extremely good dispersibility in an aqueous dispersion medium having a pH of 3 to 9.

  As described above, the dispersion particle diameter of the coated composite particle containing a hydrophilic polymer having a carboxyl group or a hydrophilic polymer that is an amine is stabilized at 50 nm or more and 200 nm or less under physiological conditions. It exhibits excellent dispersion stability and fluidity in the bloodstream of mammals, and functions effectively as a kind of DDS system. Then, cell destruction in the specific tissue can be promoted by irradiating the composite particles delivered to the specific tissue such as the target liver tissue by the DDS system with ultrasonic waves from outside the living body.

  In the present embodiment, for example, in order to form a hydrophilic polymer film having a plurality of carboxyl groups on titanium dioxide fine particles, titanium dioxide fine particles and a hydrophilic polymer having a plurality of carboxyl groups are dispersed in dimethylformamide to form 90- What is necessary is just to perform a hydrothermal reaction at 180 degreeC for 1 to 12 hours, and to combine both by an ester bond. 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 the absorption band of the ester bond by infrared spectroscopy.

  In this embodiment, for example, in order to form a hydrophilic polymer film containing amine on titanium dioxide fine particles, a mixture of titanium dioxide fine particles and hydrophilic polymer containing amine dispersed in dimethylformamide is heated and bonded. The reaction may be carried out. At this time, if the ratio of titanium dioxide and hydrophilic polymer is appropriately selected, the reaction proceeds even without pressurization. However, since the reaction is further promoted when pressure is applied, it is desirable to proceed the reaction under pressure. In this case, when polyethyleneimine (average molecular weight: 10,000) is used as the hydrophilic polymer, the final concentration of polyethyleneimine is preferably 10 mg / ml or more in order to make the dispersibility more suitable. In this manufacturing method, the heating temperature is preferably 80 to 220 ° C.

  Thus, in this embodiment, since the polymer film is formed on the surface of the base particle, the carboxyl residue or amino group of the hydrophilic polymer contained in the polymer film is specific to the target molecule. Molecules having binding ability can be immobilized. For example, for the immobilization of molecules having specific binding ability to the polymer film on the surface of the substrate particles, an amino coupling method is mainly performed using amino groups or carboxyl groups of the molecules.

  For example, an amino group in a polypeptide that specifically recognizes a specific biological tissue and a carboxyl group of the polymer coating are covalently bonded by a dehydration condensation reaction, and the specific biological tissue is attached to the polymer coating on the surface of the substrate particle. Polypeptides that specifically recognize can be bound. Alternatively, the amino group of the polymer coating and the carboxyl group in the polypeptide that specifically recognizes the specific biological tissue are covalently bonded by a dehydration condensation reaction, and the specific biological tissue is attached to the polymer coating on the surface of the substrate particle. A polypeptide that specifically recognizes can also be bound.

  As mentioned above, although embodiment of this invention was described with reference to drawings, these are the illustrations of this invention, Various structures other than the above are also employable.

  For example, in the above embodiment, the polypeptide that specifically recognizes a specific biological tissue is a polypeptide that specifically recognizes the liver tissue of a mammal, but other tissues may be targeted. For example, mammalian epithelial tissue, supporting tissue, muscle tissue, nerve tissue, vascular tissue, tissues of various organs other than the liver, and the like may be targeted. In this way, an excellent DDS can be similarly constructed for various tissues other than liver tissues by using polypeptides that specifically recognize these tissues.

  In the above embodiment, the preS1 / S2 sequence is used as the amino acid sequence derived from the surface antigen of hepatitis B virus, but other sequences may be used. For example, a sequence having 80% or more homology with the hepatitis B virus preS1 / S2 sequence or an amino acid sequence obtained by deleting, substituting, or adding one or several amino acids of the hepatitis B virus preS1 / S2 sequence Even if it is used, it has a liver tissue recognition ability substantially equivalent to that of the preS1 / S2 sequence, so that it is possible to construct an excellent DDS for liver tissue.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

The purpose of this study was to use TiO ₂ nanoparticles immobilized with a protein derived from hepatitis B virus, in vitro incorporation into cultured cancer cells and TiO ₂ / U. S. This includes studying the mechanism of cancer cell damage by law.

Therefore, in this study, we examined as follows.
Example 1: The experimental reagents and apparatus used in this study will be described.
Example 2: The purification of hepatocyte recognition protein preS1 / S2 is shown.
Example 3: shows the results for the immobilization of preSl / S2 protein TiO ₂ above.
Example 4: Results are shown for functional evaluation of preS1 / S2 immobilized TiO ₂ using an SPR sensor.
Example 5: Results are shown for the targeting ability of preS1 / S2 protein-immobilized TiO ₂ to hepatocytes using immunostaining.
Example 6: _TiO 2 / U. S. The radical purification by the method was evaluated using a fluorescent probe reagent.
Example 7: preS1 / S2 _TiO using immobilized TiO _{2 2} / U. S. The result of the cell damage effect by the method is shown.

<Example 1: Experimental reagents and apparatus>
[Tumor cells]
In normal cells, regulatory mechanisms are working so that they do not increase too much. However, when cells become cancerous, this regulatory mechanism does not work well and continues to divide indefinitely.

  Cancer is classified according to the tissue or cell type from which it is derived. Cancers that originate from epithelial cells are called carcinomas, those that originate from connective tissue or muscle cells are called sarcomas, and leukemias that originate from hematopoietic cells in cancers that do not fall into either of these two categories. There are also cancers derived from cells of the nervous system.

HepG2
In this study, human liver cancer-derived cancer cells (HepG2) were used. In this experiment, all cells in the logarithmic growth phase were used. This cell was provided by Associate Professor Shunichi Kuroda, Institute of Scientific and Industrial Research, Osaka University.

Culturing method The medium is DMEM + 10% FBS + 60 mg / ml Penicillin + 100 mg / ml Streptomycin, and is cultured in an incubator containing 37 ° C. and 5% CO ₂ . The subculture method of the cells was carried out according to the following procedure, and when the subculture was performed 10 times (about 1 month), the cells were discarded and new cryopreserved cells were used.
(1) Remove the medium and wash with PBS (−) (2) Add 2.5 g / L Trypsin + 1 mM EDTA-2Na, and incubate in an incubator (37 ° C., 5% CO ₂ ) for 2-3 min.
(3) Add DMEM to neutralize Trypsin and detach all cells by pipetting.
(4) Centrifuge (1000 rpm, 5 min), remove the supernatant and add fresh medium.
(5) Adjust to the desired cell concentration and seed in a new dish.

[titanium dioxide]
Polyacrylic acid modified titanium (PA-TiO ₂ )
The isoelectric point of TiO ₂ is around pH 6.2 and aggregates when suspended in the environment and medium (pH 7.4). In this study to enhance the dispersibility of TiO _2, using titanium dioxide having a modified polyacrylic acid TiO ₂ surface (PA-TiO _2). In the vicinity of neutrality, PA-TiO ₂ is charged with a negative charge and is dispersed by electrostatic repulsion. The average particle diameter measured by HPPS is about 90 nm (FIG. 7-1 described later).

PA-TiO ₂ preparation method (1) DMF is added to polyacrylic acid (PAA) and mixed to obtain a 100 mg / ml PAA solution.
(2) 10 ml of TiO ₂ suspension and 100 mg / ml PAA solution of (1) were put into 50 ml Corning, and subjected to ultrasonic irradiation with an ultrasonic generator for 30 minutes.
(3) Mix DMF 37.5 ml → PAA solution 2 ml → TiO ₂ suspension 0.5 ml in this order using 50 ml Corning.
(4) Sonicate the mixture of (3) until there is no precipitation.
(5) Place in a Teflon sealed container and react at 150 ° C. for 5 hours (start the evening reaction and leave until the next day).
(6) Divide the reaction liquid into 10-ml portions of 50 ml Corning.
(7) After adding and stirring twice (20 ml) of acetone to (6), the mixture was allowed to stand at room temperature for 1 hour.
(8) Centrifuge at 4000 rpm, 15 ° C., 20 min in a centrifuge.
(9) Discard the separated supernatant by decantation so that the precipitate does not come off.
(10) Add 99% Ethanol twice (20 ml) and mix gently.
(11) Centrifuge at 4000 rpm for 10 minutes in a centrifuge.
(12) After the separated supernatant is discarded by decantation so that the precipitate does not come off, W. Suspend to the required concentration.

[reagent]
Cell culture , Dulbecco's Modified Eagle Medium (DMEM) (Nacalai Tesque)
・ Foetal Bovine Serum (FBS) (GIBCO)
・ PBS (-) (Nissui Pharmaceutical Co., Ltd.)
・ Typsin (Sigma)
EDTA-2Na (Ethylenediaminetic acid-2Na-2H ₂ O) (Nacalai Tesque)

ATP assay
CellTiter-Glo Luminescent Cell Viability Assay (G7570) (Promega)

LDH assay
CytoTox-ONE Homogenous Membrane Integrity Assay (G7890) (Promega)

Trypan blue staining method / Trypan blue (BECKMAN COULTER)

Radical measurement / Aminophenyl Fluorescein (APF) (Daiichi Chemical)

Antibody・ Anti-preS1 monoantibody ・ Anti-preS2 monoantibody (Special Immunology Institute)

・ Anti-GFP monoclonal antibody (Nacalai Tesque)
Alexa Floor 555 goat anti-mouse IgG antibody (Invitrogen)

Reagent / Tryptone
・ Yeast Extract
・ NaCl
・ Agarose
・ Ampicillin
・ Isopropyl-thio-β-D-galactoside (IPTG)
・ Triton X-100
・ Urea ・ Dithiothreitol (DTT)
・ 2-ethanolamine
・ Dimethylformamide (DMF)
・ Bovin Serum Albumin (BSA) (Nacalai Tesque)
・ Phenylmethylsulfuryl fluoride (PMSF)
・ 4 ', 6-diamidino-2-phenyllinole (DAPI) (SIGMA)
1-Ethyl-3- (3-dimethylaminopropyl) -carbohydrate, hydrochloride (EDC)
・ N-Hydroxysuccinimide (NHS)
・ Polyacrylamide (PAA)
・ Paraholmaldehyde (PFA) (Wako Pure Chemical Industries)

Titanium dioxide MTT-422 (Ishihara Sangyo)

Gel filtration chromatography carrier Sephadex G-50
・ Sephacryl S-500 HR
・ PD-10 Column (GE Healthcare Bioscience)

Dialysis membrane , Dialysis Membrane, Size 20 (Wako Pure Chemical Industries)

Strains BL21 (DE3) (F-ompT hsdSB (rB-mB-) gal dcm (DE3) (Novagen)

Plasmid pGEX-GFP-preS1 / S2 (FIG. 6)
・ PGEX-GFP (Institute for Industrial Science, Osaka University)

  In this study, protein expression was performed using a plasmid in which the GST, GFP, and preS1 / S2 genes were incorporated. GST is a carrier protein that is a tag for affinity purification, and GFP is a fluorescent protein that develops green fluorescence. PreS1 / S2 is a molecular recognition protein that recognizes hepatocytes.

[Buffers and media]
The buffer and medium used in this study were prepared as follows.

LB medium Tripton: 1.0 g
Yeast Extract: 0.5g
NaCl: 0.5g
These are dissolved in 100 ml of distilled water and then autoclaved. Allow to cool before adding 100 ml of ampicillin.

1M Hepes buffer (pH 7.4)
HEPES: 238.9g
This is dissolved in ultrapure water and then adjusted to pH 7.4 with 4N NaOH. Thereafter, the volume is increased to 1 L, sealed and autoclaved. Moreover, it created with the same operation also when producing Hepes buffer of pH 8.0.

Blocking buffer
FBS: 1ml
BSA: 1.5g
Triton X-100: 15ml
These are dissolved in PBA (−) and adjusted to 50 ml. Dispense and store frozen.

Inclusion body washing liquid 1M Hepes buffer (pH 8.0): 4 ml
10% Triton X-100: 10ml
0.5M EDTA: 0.4ml
After dissolving in ultrapure water, make up to 200 ml.

8M urea solution urea: 96g
1M Hepes buffer (pH 8.0): 4 ml
1 mM DTT: 0.2 ml
0.5M EDTA: 0.4ml
After dissolving in ultrapure water, make up to 200 ml.

4M urea solution urea: 240g
1M Hepes buffer (pH 8.0): 20 ml
1 mM DTT: 1 ml
0.5 mM EDTA: 2 ml
After dissolving in ultrapure water, make up to 1 L.

2M urea solution urea: 120g
1M Hepes buffer (pH 8.0): 20 ml
1 mM DTT: 1 ml
1 mM EDTA: 2 ml
After dissolving in ultrapure water, make up to 1 L.

Separating Gel (SDS-PAGE) (12.5%)
D. W. : 2.0ml
Sol. A (1.5 M Tris-HCl (pH 8.8)): 1.5 ml
Sol. C (30% Acrylamide, 0.8% Bisacrylamide): 2.5 ml
10% SDS: 60 ml
10% ammonium persulfate (APS): 80ml
TEMED: 10ml
Mix with the above composition. APS is added last.

Stacking Gel (SDS-PAGE) (3.5%)
D. W. : 2.75ml
Sol. B (0.5 M Tris-HCl (pH 6.8)): 0.75 ml
Sol. C (30% Acrylamide, 0.8% Bisacrylamide): 0.7 ml
10% SDS: 50 ml
10% ammonium persulfate (APS): 50 ml
TEMED: 5ml
Mix with the above composition. APS is added last.

× 10 SDS-PAGE running buffer Tris: 30.3 g
Glycine: 143.1g
SDS: 10g
D. W. After dissolving in 1 ml, make up to 1 liter.

× 2 SDS sample buffer
0.5M Tris-HCl (pH 6.8): 25 ml
10% SDS: 40 ml
Glycerol: 20ml
Bromophenol Blue (BPB): 10mg
Adjust with the above composition.

[apparatus]
CO2 incubator APC-30D (ASTEC)

Safety cabinet LAD-1300XA Class II (Oriental Giken Kogyo)

Ultrasonic irradiation equipment・ SONIC MASTER ES-2 (OG GIKEN)

Centrifuge / KUBOTA 6930 (KUBOTA)
Cetrifuge 5415 R (eppendorf)

Luminometer / Veritas Microplate Luminometer (Promega)

Autoclave High-Pressure Steam Stylizer BS-305 (TOMY)
・ KT-2346 (ALP)

Oven ST0-82 (IWAKI)

Fluorescent microplate reader CYTOFLUOR Multi-well Plate Reader Series 4000 (Per Septive Biosystems)

Spectrophotometer · Spectrophotometer U-3010 (HITACHI)

Flow cytometry EPICS XL-MCL ADC (BECKMAN COULTER)

SPR sensor BIACORE2000 (BIACORE)

Microscope / Universal inverted microscope Diaphoto TMD300
・ Microscope VSTB-2 (Nikon)
・ Fluorescence microscope Bio zero (BZ-8000) (Keyence)

FPLC
・ AKTA (GE Healthcare Bioscience)

HPPS
・ High Performance Particle Sizer (MALVERN)
The principle of HPPS is that most dynamic light scattering (DLS) system manufacturers irradiate the particles with laser light and analyze the fluctuations in the intensity of the scattered light at a 90 degree position. A method that relates Brownian motion to particle size is used. The system used this time measures so-called backscattered light close to 180 degrees (173 degrees). Brownian motion is an irregular motion of particles caused by random collisions of liquid molecules around the particles. It is characterized by small particles moving fast and large particles moving slowly. As a result of the particles moving in this way, the light intensity of the laser light fluctuates, and the particle size is calculated by measuring the fluctuation speed. Incidentally, the results of measuring the particle size distribution of the above PA-TiO ₂ particles in Fig.

[Equipment]
Cell culture dish (3000-035)
Cell culture dish (4000-010)
Cell culture dish (4020-010) (IWAKI)
・ 10ml pipette (356551) (FALCON)
・ 24-hole plate (3526)
・ 96-well plate (3603) (Costar)
・ 0.22mm filter (SLGV J13SL) (Millex)

<Example 2: Purification of preS1 / S2 and GFP protein>
[Introduction]
In preparing hepatocyte targeting TiO ₂ , the purification of hepatitis B virus-derived preS1 / S2 protein having hepatocyte specificity was first examined. The preS1 / S2 protein used this time was expressed by the following procedure using Escherichia coli as a fusion protein with the fluorescent protein GST-GFP.

[experimental method]
The following experimental method was performed with reference to Shigeo Ohno, Yoshifumi Nishimura: Protein Experiment Protocol 1 Functional Analysis (1997) Shujunsha.

Protein Expression (1) Add 1 ml of pGEX-hGFP-preS1 / S2 and 1 ml of pGEX-hGFP plasmid to 100 ml of E. coli BL21 (DE3).
(2) After leaving on ice for 30 minutes, add heat shock at 42 ° C. for 60 seconds.
(3) After cooling on ice for 5 minutes, seed on LB plate (+ Amp) and incubate overnight at 37 ° C.
(4) The colonies on the plate are inoculated into 3 ml of LB medium (+ Amp) with a toothpick and cultured at 37 ° C. for 8 hours.
(5) Add 1 ml of E. coli cultured with 200 ml of LB medium (+ Amp) to an Erlenmeyer flask and incubate at 37 ° C.
(6) The culture solution is O. D. When 600 = 0.8 to 1.0, 200 ml of 100 mM IPTG is added and cultured at 17 ° C. for 12 hours.

Protein recovery (1) Transfer 200 ml of the culture solution to a centrifuge tube, collect it by centrifugation at 10,000 rpm for 10 minutes at 4 ° C.
(2) Remove the supernatant, add 30 ml of 20 mM Hepes buffer (pH 8.0), and suspend the cells.
(3) Transfer the solution to Corning and add 300 ml of 10 mg / ml PMSF.
(4) Ultrasound is applied (OUTPUT 5, DUTY 40, 5 minutes) to disrupt E. coli.
(5) Centrifuge at 10000 rpm for 10 minutes at 4 ° C. to collect the supernatant.

Purification of protein from insoluble fraction (1) Add 10 ml of washing solution of inclusion body to the insoluble fraction (precipitate) and suspend.
(2) Centrifuge at 10000 rpm for 10 minutes at 4 ° C.
(3) Repeat operations (1) and (2) once more.
(4) Add 10 ml of 8M urea solution to the precipitate, suspend well, and let stand at room temperature for 1 hour.
(5) Centrifuge at 10000 rpm for 10 minutes at 4 ° C.
(6) The supernatant is transferred to a dialysis tube and dialyzed against 500 ml of 4M urea solution at 4 ° C. for 2.5 hours.
(7) Replace the dialyzed external solution with 500 ml of 2M urea solution and dialyze at 4 ° C. for 2.5 hours.
(8) Exchange the dialyzed external solution to 20 mM Hepes buffer (pH 8.0) and dialyze overnight at 4 ° C.
(9) Remove the solution from the dialysis tube and centrifuge at 10000 rpm for 10 minutes at 4 ° C. to collect the supernatant.

[Protein evaluation]
Determination of protein by Bradford method The Bradford method was used as a method for measuring the protein concentration in the waste liquid. The Bradford method uses the fact that Coomassie Brilliant Blue (CBB) in the dye solution binds to the protein in the sample and shifts the maximum absorption wavelength from 465 nm to 595 nm. By measuring this wavelength with an absorptiometer, the protein concentration is measured. It is a method to do.

The measurement was performed according to the following procedure.
(1) Take 800 ml of the sample solution diluted to the measurable range in an Eppendorf tube. The standard BSA solution for the calibration curve is adjusted to 0, 1, 5, 10, 20 mg / ml.
(2) Add 200 μl of the dye solution CBB solution and stir well.
(3) Measure within 1 hour with a spectrophotometer (wavelength: 595 nm).
(4) Draw a calibration curve and calculate the protein concentration.

SDS-PAGE
Preparation of sample The sample was prepared by the following method.
(1) Mix 2-mercaptoethanol and 2 × SDS Sample Buffer 1: 9 (use 15 ml per sample).
(2) The sample protein solution and the one made in step 1 are mixed 1: 1 (1 sample 15 ml: mixed at 15 ml).
(3) Heat treatment at 95 ° C. for 3 minutes.
(4) Leave on ice for at least 5 minutes.
(5) Inject 20 ml of Sample into the gel spot.

Detection of protein by CBB staining (1) After completion of SDS-PAGE, the gel is taken out, unnecessary portions are cut off, transferred to a container, and a staining solution of about 5 to 10 times the gel volume is added and gently shaken for 1 hour.
(2) Excluding the staining solution, pour a decolorizing solution of about 5 to 10 times the gel volume, and shake gently until decolorization of the gel is complete and a protein band is clearly recognized.
(3) Remove the gel.

Protein quantification Gels are loaded with a scanner and analyzed with analysis software (Scion Image). From the image captured by the scanner, the density of the band is digitized (Measure), and compared to the standard BSA solution (25, 50, 100 mg / ml) whose concentration is determined, presume.

[Results and discussion]
GST-GFP-preS1 / S2 and GST-GFP proteins were purified, and the proteins were quantified by Bradford method and SDS-PAGE (FIG. 8, Table 1).

  It is desirable to purify the protein from the soluble fraction (U.S. fine powder after disruption), but when observing the solution obtained from this soluble fraction, almost no GFP fluorescence was observed in both proteins. . Therefore, a method of extracting protein from inclusion bodies of insoluble fraction was used. The protein obtained by this method shows deep green fluorescence, and it is considered that a high concentration of GST-GFP-preS1 / S2 or GST-GFP protein could be purified. When these protein concentrations were determined by the Bradford method, preS1 / S2 was obtained at a concentration of 2.98 mg / ml. However, since it is considered that these protein solutions contain a lot of impure proteins other than preS1 / S2, confirmation of the molecular weight of the protein purified by SDS-PAGE method and concentration measurement by analysis software were also performed.

  From this result, it is known that the molecular weight of GST-GFP-preS1 / S2 is about 72 kDa, and that of GST-GFP is about 54 kDa. From FIG. 8, since bands appeared at the respective molecular weight positions, it was confirmed that the GST-GFP-preS1 / S2 and GST-GFP proteins were purified.

  Further, from the supernatant solution, the preS1 / S2 protein had a concentration of only 0.048 mg / ml, and there were many bands at different molecular weight positions. However, in the purification from inclusion bodies, a high concentration of 1.79 mg / ml protein is obtained, and compared to the SDS gel in the supernatant, no band is seen at other molecular weight positions and a good purity is obtained. I was able to. Similar results were seen with GST-GFP protein. Therefore, protein will be expressed in this way in the future, and the protein purified from inclusion bodies will be used.

<Example 3: Immobilization of preS1 / S2 and GFP protein on nanoparticle surface>
[Introduction]
In Example 2, the preS1 / S2 protein could be purified. In this example, protein immobilization on the surface of PA-TiO ₂ using the amino coupling method is examined, and separation and purification of preS1 / S2 immobilized particles using gel filtration chromatography are also performed.

[Principle of gel filtration chromatography]
As a method of separating proteins,
l Gel filtration chromatography
l Ion exchange chromatography
l (Antibody) Affinity chromatography is used, but gel filtration chromatography was used in this study.

  In principle, proteins with different masses can be separated by gel filtration chromatography using porous beads such as polyacrylamide gel, dextran (bacterial polysaccharide), or agarose (seaweed polysaccharide). When the protein solution is flowed through a column packed with beads, the protein stays in the pores of the beads for some time while flowing around the beads. As a result, the movement of this protein within the column is slow. Smaller proteins can easily penetrate into the pores of the beads and flow more slowly through the column than larger proteins (Figure 9).

[experimental method]
Protein immobilization method (Figure 10)
(1) Add 0.25 ml of 0.1 M NHS solution to 0.25 ml of 0.4 M EDC solution.
(2) 0.5 ml of the mixture of (1) is added to 2.5 ml of 1.5% PA-TiO ₂ solution, and the mixture is allowed to react at room temperature for 1 hour with gentle stirring.
(3) separating the EDC / NHS unreacted with PA-TiO ₂ by gel filtration column PD-10.
(4) Add 2.5 ml of the protein solution to 3.5 ml of the PA-TiO ₂ solution coming out of the column, and react while gently stirring at 4 ° C. for 4 hours to overnight.
(5) Add 1 ml of 0.1M monoethanolamine and react at 4 ° C. with gentle stirring for 30 minutes.

Gel filtration chromatography (AKTA)
(1) Turn on the attached PC and start the program.
(2) The column is equilibrated in advance with 20 mM Hepes buffer (pH 7.4, filtration (0.22 mm)).
(3) The sample solution is injected into the 2 ml sample loop, and the program is set so that the flow rate is 0.5 ml / min and the sample is fractionated by 4 ml.

[Results and discussion]
The results of fraction purification gel filtration chromatography by gel filtration chromatography are shown in FIGS. In FIG. 11, the fraction in which the preS1 / S2 protein is present was examined by measuring the fluorescence of each fraction. In FIG. 12, the amount of protein in each fraction was examined by the Bradford method. When gel filtration only preSl / S2 protein as compared simultaneously, and also to obtain simultaneously chromatogram when only PA-TiO ₂ was gel filtered.

When these were compared, it was found that the fraction of TiO ₂ became fractions 7 to 11 based on the value of the UV detector. And it was found that the preS1 / S2 protein was fractionated into fractions 11-18. For these reasons, fractionation of preSl / S2 immobilized TiO ₂ is considered that it would be have become 7-10. The amount of protein present on the particles was 64 mg / L.

To increase the amount of immobilization,
・ Condition of amino coupling method (EDC / NHS) (N. Nakajima, Y. Ikada. Mechanism of Amide Formation by Carbohydrate for Bioconjugation in Aquamedia Med.
・ Study using other Linker reagents ・ Increase protein purity (further purification of preS1 / S2 by affinity chromatography)
It is considered that it is possible to create particles in which more proteins are modified by performing the above.

Confirmation of immobilization by SDS-PAGE Each sample fractionated by gel filtration chromatography was subjected to SDS-PAGE and peeled off or adhered to the particles by thermal denaturation, and unreacted protein was detected. (FIG. 13). As a result, a band was observed at the position of the molecular weight of the preS1 / S2 protein in fractions 8 to 16. This correlates with the data in FIG. 11 and FIG. 12, and since dark bands are obtained in the fractions 8 and 9 where TiO ₂ is detected, the future will be comprehensively viewed in FIGS. It is considered better to use fraction 8 for research.

Particle Stability Purified TiO ₂ particles were placed in DMEM containing 10% FBS, and the change in particle diameter under the same environment as in vivo was examined (FIG. 14). Usually, it is known that TiO ₂ whose surface charge is near neutral is likely to aggregate in an in vivo environment. Since PA-TiO ₂ was modified with polyacrylic acid, the dispersibility was improved by the carboxyl group on the surface. It was found that even after preS1 / S2 immobilization this time, the TiO ₂ particles had a good dispersibility with a particle size of 50 to 200 nm and some aggregation was observed, but the dispersibility was maintained after 24 and 72 hours. From these results, in an in vivo experimental system, in addition to the targeting function, it is considered that accumulation of preS1 / S2 immobilized TiO ₂ particles by the EPR effect in cancer tissues can be expected.

[Summary]
It was found that the preS1 / S2 protein was bound on the TiO ₂ particles by the protein immobilization by the present amino coupling method. It was also found that the amount of protein was 64 mg / L, and the average particle size was about 100 nm. This means that three preS1 / S2 molecules are bonded on average per particle.

  It was also found that the particles prepared this time hardly aggregate even in 10% FBS and have a stable particle size. In Example 4 and later, functional evaluation of the particles was performed.

<Example 4: PreS1 / S2 immobilized TiO ₂ specificity evaluation by SPR sensor>
[Introduction]
Up to Example 3, preS1 / S2 immobilized TiO ₂ was prepared and fractionated and purified by gel filtration chromatography. In this example, an experiment for evaluating the specificity of the preS1 / S2 immobilized TiO ₂ against the anti-preS1 antibody is performed on the completed preS1 / S2 immobilized TiO ₂ using an SPR sensor.

[Detection principle of SPR sensor (BIACORE)]
The BIACORE detection system employs an optical phenomenon of so-called surface plasmon resonance (SPR) that occurs when surface plasmons are excited at the metal / liquid interface. A light-emitting diode is used as a light source, and polarized light having a wavelength of 760 nm is irradiated onto the prism, condensed into wedge-shaped light on the prism, and irradiated onto the sensor chip under conditions of total reflection. When this light is totally reflected, a special wave is generated on the gold film surface. Since some of the wavelength energy of the incident light is consumed to generate the wave, when the reflected light is monitored, the energy disappears at a specific angle, and when the intensity of the reflected light over the entire wavelength range is measured, A valley of light with reduced intensity can be confirmed at an angle. This decrease is called SPR. The disappearance angle (SPR angle) of the reflected light varies depending on the refractive index of the medium in the vicinity of the gold film surface. Using this decrease, the binding / dissociation between two molecules is measured. By immobilizing the sample (ligand) on the gold thin film and injecting a specific substance (analyte) to it, the two molecules bind to each other, increasing the mass of the gold thin film, resulting in the sensor chip surface. The refractive index of increases. The valley of light moves by this change in refractive index (FIG. 15). By displaying this change in mobility over time in a graph called a sensorgram, the interaction between molecules can be monitored in real time.

As a unit representing the mobility of the valley of light, a change of 0.1 ° in the SPR angle is defined as 1000 resonance units (RU). 1000 RU resulted in a mass change of about 1 ng / mm ^{2 on} the sensor chip surface.

[Immobilization of anti-preS1 antibody to sensor chip]
In BIACORE, the sensor chip is always in contact with the fluid during measurement, and the obtained sensorgram directly reflects the refractive index change of the solvent on the surface of the sensor chip. Therefore, the change in the RU value is the sum of the sensorgram resulting from the binding between the ligand immobilized on the sensor chip and the analyte in the solvent and the refractive index of the solvent itself, and the influence of the solvent at this time Is called the bulk effect.

  A typical sensorgram for BIACORE is shown in FIG. When an analyte is injected and added to the surface of a sensor chip on which a ligand is immobilized, the resonance sensorgram increases with the specific binding reaction. After the set time, the injection of the analyte is completed and the running buffer starts to flow. Here, the dissociation reaction of the analyte once bound to the ligand is observed. In order to separate molecules that are strongly bonded, a denaturant such as a surfactant is added to completely dissociate the analyte and the ligand and regenerate the sensor chip. By repeating this cycle, it is possible to process many samples continuously. In addition, since the solution, sample, and the like used in BIACORE are passed through the microchannel, it is necessary to perform degassing sufficiently by performing a filtration process in order to avoid mixing of fine particles.

  In this study, the preS1 / S2 protein is immobilized on the gold thin film of the sensor chip by an amino coupling reaction. An amino coupling chip CM was used as the sensor chip. There is a hydrophilic dextran layer on the gold thin film surface of CM5, and this dextran is modified with a carboxyl group. The carboxyl group and the amino group of the anti-preS1 antibody were used for immobilization by coupling.

Thus, the anti-preS1 antibody was used as a ligand, and preS1 / S2 immobilized TiO ₂ prepared up to Example 3 was used as an analyte. As the running buffer, 50 mM Hepes buffer (pH 7.4, filtration (0.22 mm)) was used.

[Specificity evaluation of preS1 / S2 protein by BIACORE]
Evaluation of concentration dependency by preS1 / S2 protein Anti-preS1 antibody was immobilized on a sensor chip (CM5) using a program, and 20 ml of 1,7,14 mM preS1 / S2 protein was flowed there, and concentration dependency in this antibody Sex was evaluated. Then, the sensor chip was regenerated by finally dissociating in 10 mM NaOH + 10% SDS.

preS1 / S2 immobilized specificity by TiO ₂ Evaluation sensor chip immobilized anti preS1 on the antibody on (CM5) preS1 / S2 immobilized TiO ₂ flushed 20 ml, and assess the specificity of the anti-preS1 antibodies. Then, the sensor chip was regenerated by finally dissociating in 10 mM NaOH + 10% SDS.

[Results and discussion]
The concentration dependency of the preS1 / S2 protein by the anti-preS1 antibody and the specificity of the preS1 / S2 immobilized TiO ₂ were evaluated (FIGS. 17 and 18). As a result of flowing 1, 7, and 14 mM preS1 / S2 protein, 370 RU was obtained at 14 mM, and 240 RU was obtained at 7 mM. As the protein concentration was lowered, a clean concentration-dependent sensorgram could be obtained. However, with 1 mM preS1 / S2 protein, a specific reaction with an antibody was hardly obtained at about 20 RU (FIG. 17). In addition, when preS1 / S2 immobilized TiO ₂ was allowed to flow, BSA-immobilized TiO ₂ and TiO ₂ showed almost no reaction with antibodies. On the other hand, preS1 / S2 immobilized TiO ₂ showed a large specificity of about 80 RU, despite the amount of protein being 1 mM or less from the previous FIG. 17 with 0.3 mM protein in the solution (FIG. 18). This is because the value of RU increases due to the change in the mass of the reacted substance, so it does not mean that many proteins have reacted. The mass of TiO ₂ is considerably larger than that of protein, so it is 80 RU with 0.3 mM protein. It is thought that the value was obtained. Therefore, the preS1 / S2 immobilized TiO ₂ of this study has the specificity of preS1 / S2, and from the BIACORE data, it can be shown again that the preS1 / S2 protein is immobilized on the TiO ₂ surface.

<Example 5: Immunostaining method>
[Introduction]
In Example 4, the specificity to the antibody was shown by using the SPR sensor, and at the same time, it was confirmed that the preS1 / S2 protein was immobilized on the TiO ₂ surface. In this example, human liver cancer-derived HepG2 cultured cells were actually used to evaluate hepatocyte specificity by immunostaining.

[principle]
The following immunostaining method is described by Hiroshi Konan, Toshio Kuroki, edited by Experimental Medicine, Separately, Cultured Cell Experiment Handbook (2004) Yodosha, and Ricci, M .; Bertolotti, C.I. Anzivino, et al. 17b-estradiol presents cytologicality hydrophobic bill acid in HepG2 and WRL-68 cell cultures. J. et al. Gastroenterology and Hepatology 21 (2006) 894-901.

  First, cells are cultured on equipment suitable for observation after culture and staining. After appropriate fixation, the cell membrane is permeabilized so that the antibody molecules can enter the cell. Thereafter, an antibody against the molecule to be studied (primary antibody) is reacted. After the reaction with the primary antibody, the unreacted antibody is removed. A secondary antibody to which a fluorescent substance or enzyme is bound is reacted, then washed with a buffer, and observed under a fluorescence microscope (FIG. 19). After staining, nuclear staining is also performed as necessary. The enzyme here is Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP). This time, an antibody bound with Alexa Floor 555, which is a fluorescent dye, was used.

[Immunostaining with anti-preS1 / anti-GFP antibody]
Experimental method The immunostaining method is briefly shown (FIG. 20).
(1) Put 2 ml of cell suspension in a 3.5 cm dish so that it becomes 2 × 10 ⁵ cells / well.
(2) Incubate at 37 ° C., 5% CO _{2 for} 24 hours.
(3) Add 200 ml of 0.1% preS1 / S2 immobilized TiO ₂ and incubate at 37 ° C., 5% CO _{2 for} another 6 hours.
(4) Discard the medium and wash 3 times with PBS (-).
(5) Add 500 ml of 4% PFA and let stand for 15 minutes.
(6) Remove PFA, add 500 ml of PBS (−) containing 0.25% Triton X-100, and stir for 15 minutes.
(7) Add 500 ml of 1 mg / ml anti-preS1 mouse monoclonal antibody or 4.4 mg / ml anti-GFP mouse monoclonal antibody overnight.
(8) Wash 3 times with PBS (-) containing 0.03% Triton X-100.
(9) Add 500 ml of 4 mg / ml Alexa Floor 555 goat anti-mouse IgG antibody, shield from light and let stand at room temperature for 1 hour.
(10) Wash three times with PBS (-) containing 0.03% Triton X-100 (light-shielded).
(11) Add 500 ml of PBS (−) to prevent drying and observe with a fluorescence microscope.

Results and Discussion The target ability of preS1 / S2 immobilized TiO ₂ was examined by immunostaining using anti-preS1 antibody and anti-GFP antibody (FIGS. 21 and 22). The cells to which preS1 / S2 immobilized TiO ₂ was added showed strong red fluorescence by the immunostaining method specifically used for hepatocytes when either antibody was used. In addition, in the preS1 / S2 protein added as a positive control, both antibodies showed red fluorescence and were specifically taken up by hepatocytes. Here, when FIG. 22A and FIG. 22B were compared, a difference was seen in the fluorescence. With preS1 / S2 immobilized TiO ₂ , fluorescence was mainly observed around the cell membrane. On the other hand, in the preS1 / S2 protein, punctate fluorescence is seen, which seems to be taken up into the cytoplasm. Thus, with the uptake time of 6 hours, preS1 / S2 immobilized TiO ₂ specifically targeted hepatocytes but did not appear to be taken up into the cells by endocytosis.

<Example 6: Radical measurement>
[Introduction]
So far, the targeting ability of preS1 / S2 immobilized TiO ₂ has been demonstrated. TiO ₂ / U. S. The cancer cell killing effect by this method is considered to be caused by oxidation by radicals generated by irradiating TiO ₂ with ultrasonic waves. Therefore, in this chapter, TiO ₂ / U. S. The reactive oxygen species generated by the method were quantified using Aminophenyl Fluorescein (APF). This time, radical production was confirmed using Ishihara Sangyo's titanium dioxide (MPT-422).

[principle]
APF has little fluorescence in a neutral aqueous solution, but when these probes react with active oxygen species (especially .OH, .O ₂ ⁻ ) having a strong activity, fluorescein, which is a highly fluorescent compound, is generated. An increase in fluorescence intensity is observed (FIG. 23).

[Change of radical amount with ultrasonic irradiation time]
Experimental Method (1) Dilute APF to 1 mM using PBS (-).
(2) Add (1) to the 3.5 cm dish in 2 ml / well increments.
(3) adding 0.1% PA-TiO ₂ by 200 ml / well.
(4) Ultrasound (1 MHz, 30 seconds, 50% pulse) is irradiated at an output of 0 to 0.8 W / cm ² . As an irradiation method, a gel is applied to the probe portion of the ultrasonic irradiator, and the dish is placed on the gel. An ultrasonic irradiation apparatus is simply shown (FIG. 24).
(5) The amount of generated radicals was measured as fluorescence (Ex = 490 nm, Em = 515 nm).

[Results and discussion]
TiO ₂ / U. S. The effect of the amount of radicals generated by the method on the TiO ₂ concentration was examined (FIG. 25). Even when TiO ₂ was 0%, radical generation was confirmed under the present conditions, but when TiO ₂ was added and as the TiO ₂ concentration further increased, the amount of radicals generated increased. From this, TiO ₂ / U. S. It can be considered that the amount of radicals generated by the method tends to increase depending on the TiO ₂ concentration. However, when 0.1% PA-TiO ₂ was used, no significant difference was observed when compared with 0.05%. This is considered that if the TiO ₂ concentration is too high, the ultrasonic wave is blocked and the overall effect cannot be obtained.

[Summary]
TiO ₂ / U. S. By the method, it was found that radicals were also generated in the TiO ₂ particles used this time. However, under this condition, U.S. S. It can be seen that radicals are generated even in the case of only, and there is a difference in the presence or absence of TiO ₂ , but this is not a big difference, and it may be necessary to reexamine the irradiation conditions in the future.

<Example 7: Cell damage examination by TiO ₂ / US method>
[Introduction]
Up to Example 6, the specificity of preS1 / S2 immobilized TiO ₂ to antibodies and hepatoma cells was shown. In this chapter, TiO ₂ / U. S. The effect of cancer cell killing / damaging was investigated. The evaluation system used was a method for measuring the amount of lactate dehydrogenase (LDH) leaked from the cell due to damage.

[Principle of LDH]
In almost all necrosis and many apoptosis (especially late stage), molecules in the cytoplasm are leaked out of the cell due to the breakdown of the cell membrane accompanying cell death. Thus, quantitative measurement of such molecules makes it possible to quantify cells that have lost their cell membranes or lost their survival activity. Since this lactate dehydrogenase (LDH) is abundant in the cytoplasm and is a relatively stable enzyme, it is often measured for such a purpose.

  LDH is a glycolytic enzyme with a molecular weight of about 150 kDa. It is an enzyme that oxidizes lactic acid to pyruvic acid by the catalyst of coenzyme NAD +. When this is leaked from a cell damaged in the cell membrane, resazurin / diaphorase conjugate It can be quantified as fluorescence of resorufin generated through the system (FIG. 26).

The measurement method is as follows.
(1) Stabilize Substrate Mix and Assay Buffer at 22 ° C.
(2) Add 11 ml of Assay Buffer to Substrate Mix and mix gently by inversion.
(3) The cell supernatant used as a sample is seeded at 100 ml / well in a 96-well plate.
(4) Add (2) to (3) in 100 ml / well increments.
(5) Stir the plate for 30 seconds.
(6) Incubate for 10 minutes while protected from light at 22 ° C.
(7) Add 50 ml / well of Stop Solution.
(8) Stir the plate for 10 seconds.
(9) Fluorescence measurement (Ex = 560 nm, Em = 590 nm).

[Effect of ultrasonic output]
Experimental method (1) 2 ml of cell suspension is put into a 3.5 cm dish so that it becomes 2 × 10 ⁵ cells / well.
(2) Incubate at 37 ° C., 5% CO _{2 for} 24 hours.
(3) Discard the medium and add 2 ml of fresh medium.
(4) Ultrasonic conditions were 1 MHz, 50% duty ratio, irradiation for 30 seconds, and then LDH activity was measured.

[Results and discussion]
A result is shown about the influence on the cell by an ultrasonic intensity change (FIG. 27). Output increases gradually LDH values from around ^{0.2W / cm 2, 0.8W / cm} 2 or more comprising the the way to its raised has become gradual. From this result and the data of the radical-capturing fluorescent probe reagent in Example 6, U.S. S. It is considered that the threshold value of cavitation due to is about 0.3 or 0.4 W / cm ² . Therefore, as the next examination item, preS1 / S2 immobilized TiO ² is taken into hepatocytes, and then the ultrasonic damage with an output of 0.2, 0.4, and 0.6 W / cm ² is applied to give a superior cell damage effect. Let's consider.

[Effect of preS1 / S2 immobilized TiO ₂ ]
Experimental method (1) 2 ml of cell suspension is put into a 3.5 cm dish so that it becomes 2 × 10 ⁵ cells / well.
(2) Incubate at 37 ° C., 5% CO _{2 for} 24 hours.
(3) Add 200 ml of 0.1% preS1 / S2 immobilized TiO ₂ and incubate at 37 ° C., 5% CO _{2 for} another 6 hours.
(4) Discard the medium, wash 3 times with DMEM, and add 2 ml of fresh medium.
(5) Ultrasonic conditions were 1 MHz, 50% duty ratio, irradiation for 30 seconds, and then LDH activity was measured.

[Results and discussion]
preS1 / S2 immobilized TiO ₂ and TiO ₂ / U. S. The result of the cell damage effect by the method was shown (FIG. 28). Every time the output was increased from 0.2 W / cm ² , output-dependent data was obtained under any conditions. Among these, at 0.4 W / cm ² , S. And PA-TiO ² , PA-TiO ² and preS1 / S2 immobilized TiO ² were not significantly different. However, U. S. Only Comparing the preSl / S2 immobilized TiO ₂ towards the preSl / S2 immobilized TiO ₂ was superior in the cell damaging effects. On the other hand, many radicals as true from 0.2 W / cm ² in FIG. 27 does not occur, also obtaining a significant difference in either case for strong output reversed at 0.6 W / cm ² I couldn't.

[Effect of ultrasonic irradiation time]
Experimental method (1) 2 ml of cell suspension is put into a 3.5 cm dish so that it becomes 2 × 10 ⁵ cells / well.
(2) Incubate at 37 ° C., 5% CO _{2 for} 24 hours.
(3) Add 200 ml of 0.1% preS1 / S2 immobilized TiO ₂ and incubate at 37 ° C., 5% CO _{2 for} another 6 hours.
(4) Discard the medium, wash 3 times with DMEM, and add 2 ml of fresh medium.
(5) Ultrasonic conditions were 1 MHz, 50% duty ratio, 0.4 W / cm ² for 0 to 60 seconds, and then LDH activity was measured.

[Results and discussion]
The effect of the irradiation time on the cells was examined (FIG. 29). As a result, almost no difference was obtained by irradiation for 10 seconds, and on the contrary, a large LDH value was detected regardless of whether the irradiation time was too long in 60 seconds. On the other hand, in the irradiation for 30 seconds, preS1 / S2 immobilized TiO ₂ was superior in cell damage effect. Therefore, it is considered that irradiation for 30 seconds at 0.4 W / cm ² is the most effective irradiation condition.

[Summary]
Under the present irradiation conditions, it was possible to obtain data that preS1 / S2 immobilized TiO ₂ showed an excellent cell damage effect when irradiated at 0.4 W / cm ² for 30 seconds. However, in Example 6, preS1 / S2 immobilized TiO ₂ showed targeting ability specific to hepatocytes. In FIGS. 28 and 29, preS1 / S2 immobilized TiO ₂ has a strength of 0.4 W / cm ² and is irradiated with U.S. S. A significant cell damaging effect was seen as compared to only and TiO ₂ .

In the above examples 1~7, _TiO 2 / U. S. Aiming at cancer treatment using the method, the creation and functional evaluation of nanoparticles with targeting ability were performed. As a result, the following conclusions were obtained.
-The hepatocyte recognition protein preS1 / S2 was successfully immobilized by the amino coupling method. Further, this particle maintained a stable particle size without showing significant aggregation even in the same environment as that in the living body.
PreS1 / S2 immobilized TiO ₂ was confirmed to have superior specificity both in the evaluation by the SPR sensor and in the evaluation by the immunostaining method using the hepatoma cell HepG2.
· PreSl / S2 ultrasonic irradiation cytotoxic effect in the immobilized TiO ₂ presence in, U. irradiation at 0.4 W / cm ² 30 sec S. A significant cell damaging effect was seen as compared to only and TiO ₂ .

That is, the preS1 / S2 immobilized TiO ₂ produced for the first time by the present inventors is a molecular recognition protein derived from hepatitis B virus on TiO ₂ coated with polyacrylic acid as in Examples 1 to 7 above. obtained by immobilizing preS1 / S2 by the amino coupling method, having a good dispersibility at a particle size of 50 to 200 nm, and showing slight aggregation, but maintaining the dispersibility after 24 and 72 hours. .

In addition, preS1 / S2 immobilized TiO ₂ produced for the first time by the present inventors was evaluated by an SPR sensor and by an immunostaining method using hepatoma cell HepG2, as shown in Examples 1 to 7 above. In both cases, it is clear that it has specificity for confirmed hepatocytes.

Furthermore, the preS1 / S2 immobilized TiO ₂ produced for the first time by the present inventors is based on the results of examination using lactate dehydrogenase (LDH) as an index, under ultrasonic irradiation conditions at 0.4 W / cm ² for 30 seconds. It is clear that it has an excellent cytotoxic effect by irradiation.

Therefore, the preS1 / S2 immobilized TiO ₂ produced for the first time by the present inventors can apply the high reactivity of the photoexcited titanium dioxide (TiO 2) surface to the medical field. In our laboratory, it has been reported previously that hydroxyl radicals (.OH) generated when TiO ₂ is irradiated with ultrasound significantly kill cancer cells (titanium dioxide / ultrasonic catalysis ( TiO _{2 /} US method) When considered together with the results, this preS1 / S2 immobilized TiO ₂ has the effect of killing cancer cells even in vivo where light does not reach by using ultrasonic waves. Expected to be obtained.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

For example, in the above example, when preS1 / S2 immobilized TiO ₂ was irradiated with ultrasonic waves at 0.4 W / cm ² under irradiation conditions for 30 seconds, an excellent cytotoxic effect by ultrasonic irradiation was shown. Ultrasonic waves may be irradiated depending on the conditions. In this case, it is assumed that an excellent cytotoxic effect can be exhibited by appropriately adjusting the frequency (frequency), irradiation intensity, and irradiation time of the ultrasonic wave according to the treatment control, the treatment purpose, and the like. In particular, since it has an excellent cytotoxic effect on tumor cells, TiO ₂ / U. S. It may be possible to use it as a novel cancer treatment as an improved technique of the law.

It is the schematic diagram which showed the structure of the hepatitis B virus (FIG. 1a) and the hepatitis B virus envelope (FIG. 1b). TiO ₂ rutile is a schematic view showing the crystal structure of anatase type. It is a schematic view showing a photocatalyst mechanism of TiO _2. It is a schematic diagram for demonstrating the property of an ultrasonic wave. It is the model for demonstrating that the cavity which fully expanded and cannot maintain itself and reached the critical state collapses rapidly when it is drawn to the antinode part of pressure. It is a schematic diagram for demonstrating construction | assembly of a plasmid. Is a graph showing the results of measuring the particle size distribution of PA-TiO ₂ particles. It is the photograph and schematic diagram which show the result of having quantified protein by SDS-PAGE. It is a schematic diagram for demonstrating the principle of gel filtration chromatography. It is a schematic view for explaining method for immobilizing proteins to TiO ₂ particles by amino coupling method. preSl / S2 is a graph showing the results of gel filtration chromatography of proteins _{(A: TiO 2 conjugated PreS1 /} S2, B: PreS1 / S2 protein, C: PA-TiO 2). preSl / S2 is another graph showing the results of gel filtration chromatography of proteins _{(A: TiO 2 conjugated PreS1 /} S2, B: PreS1 / S2 protein, C: PA-TiO 2). Is a photograph for explaining confirm the immobilization of the PA-TiO ₂ particles of preSl / S2 protein by SDS-PAGE method. preSl / S2 protein is a graph showing the results of examining the change in the particle diameter at the same environment as in vivo immobilized PA-TiO ₂ particles. It is a schematic diagram for demonstrating the detection principle of a SPR sensor (BIACORE). It is the schematic diagram which showed the typical sensorgram of BIACORE. It is a sensorgram which shows the result of having evaluated the density | concentration dependence of the preS1 / S2 protein by an anti-preS1 antibody by BIACORE. The specificity of the preS1 / S2 immobilized TiO ₂ with anti-preS1 antibodies, another sensorgram showing the results of evaluation by BIACORE. It is a schematic diagram for demonstrating the principle of an immuno-staining method. It is the schematic diagram which showed briefly about the procedure of the immuno-staining method. Is a photograph showing the results of immunostaining with anti-PreS1 antibody _{(A: TiO 2 conjugated preS1 /} S2-GFP-GST, B: PreS1 / S2 protein, C: TiO 2 conjugated GFP-GST (scale bar = 50mm) ). Is a photograph showing the results of immunostaining with anti-GFP antibody _{(A: TiO 2 conjugated preS1 /} S2-GFP-GST, B: PreS1 / S2 protein, C: TiO 2 conjugated GFP-GST (scale bar = 50 mm ). It is a schematic diagram for demonstrating the principle of the method of quantifying a radical using APF. It is a schematic diagram which shows simply the ultrasonic irradiation apparatus. TiO ₂ / U. S. The effect of TiO ₂ concentration of radicals amount generated by the law is a graph showing the results of examining with APF. It is a schematic diagram for demonstrating that LDH can be quantified as fluorescence of resorufin produced via a resazurin / diaphorase conjugate system when leaked from a cell damaged by a cell membrane. preSl / S2 is a graph showing the results of LDH measured the effects of using immobilized TiO ₂ into cells by ultrasonic intensity changes. preSl / S2 using immobilized TiO ₂ and TiO ₂ is a graph showing the results of cell damaging effects by the ultrasonic intensity changes. preSl / S2 is a graph showing the results of LDH measured the effects of using immobilized TiO ₂ and TiO ₂ to the cells by ultrasonic wave irradiation time.

Claims (11)

Base particles that generate hydroxy radicals in an aqueous solution by ultrasonic irradiation;
A polymer coating covering at least a part of the surface of the substrate particles;
A polypeptide that binds to the polymer coating and specifically recognizes a specific biological tissue;
A composite particle comprising: The composite particle according to claim 1,
The polypeptide is a composite particle that specifically recognizes a liver tissue of a mammal. The composite particle according to claim 1 or 2,
The polypeptide is a complex particle comprising an amino acid sequence derived from a surface antigen of hepatitis B virus. In the composite particle according to any one of claims 1 to 3,
The polypeptide is
Hepatitis B virus preS1 / S2 sequence;
A sequence having 80% or more homology to the preS1 / S2 sequence of hepatitis B virus;
An amino acid sequence obtained by deleting, substituting, or adding one or several amino acids of the preS1 / S2 sequence of hepatitis B virus;
A composite particle comprising one or more amino acid sequences selected from the group consisting of: In the composite particle according to any one of claims 1 to 4,
The polypeptide is a complex particle comprising an amino acid sequence derived from a tag polypeptide. In the composite particle according to any one of claims 1 to 5,
The polypeptide is a complex particle comprising an amino acid sequence derived from a fluorescent protein. In the composite particle according to any one of claims 1 to 6,
The base particle is a composite particle that is a particle containing TiO ₂ . In the composite particle according to any one of claims 1 to 7,
The composite particle has a dispersed particle size of 50 nm or more and 200 nm or less. In the composite particle according to any one of claims 1 to 7,
The polymer film is a composite particle that is a film containing a hydrophilic polymer having a carboxyl group. The composite particle according to claim 9, wherein
The polymer film is a composite particle that is a polymer film containing polyacrylic acid. In the composite particle according to any one of claims 1 to 10,
A composite particle in which the polypeptide is bound to the polymer coating by amino coupling.