JPWO2007116808A1 - Virus particle-like structure and formulation containing the same - Google Patents

Abstract

A virus containing a metal and / or metal compound that is promising for cell-level diagnosis and treatment by incorporating a metal or metal compound effective in diagnostic imaging, tissue regeneration, or hyperthermia into a virus-like particle structure. It is an object of the present invention to provide a particle-like structure and to provide an optimal method for forming the structure. The virus particle-like structure of the present invention is a virus particle-like structure having a substantially uniform size, which contains virus proteins and fine particles containing at least one of a metal and a metal compound that are encapsulated substances. Preferably, the fine particles have a positive or negative charge as a surface charge, and contain iron, iron oxide or ferrite as a main component. It is desirable that the viral protein is one or more proteins derived from SV40 virus, JCV virus, BKV virus and / or mutants or modifications thereof.

Description

  The present invention relates to a virus particle-like structure and a preparation containing the same. The virus particle-like structure is a structure formed by organizing and including fine particles containing a metal or a metal compound in the process of self-assembly of viral proteins.

  Particles made of metal compounds are widely used in the medical field as preparations. Among them, magnetic resonance imaging (MRI), which is a technique for image diagnosis of a living body, can diagnose cancer and the like by measuring the difference in magnetic resonance relaxation time between normal tissue and diseased tissue. It is widely used as a diagnostic method without any problem. In order to make the difference in relaxation time more prominent among these, for example, oxidation described in US Pat. No. 4,827,945 (Japanese Patent Publication No. 01-500196) or International Publication No. 94/03501 pamphlet, etc. Contrast agents composed of iron particles are used in clinical settings, and these contrast agents are formulated by dispersing iron oxide particles with a polymer polysaccharide or the like.

  In addition, in the clinical field, cancer tissues are locally treated for hyperthermia using the biological property that cancer tissues are more susceptible to thermal damage than normal tissues. . This method is a method of heating living tissue by dielectric heating, and is attracting attention as a non-invasive treatment method with few side effects. However, in order to heat the whole living tissue by dielectric heating, it causes damage to normal tissue. Temperature control that does not occur is required. Therefore, as described in, for example, European Patent Application Publication No. 0444194 (Japanese Patent Laid-Open No. 2-174720) or Japanese Patent Laid-Open No. 3-128331, ferromagnetic fine particles such as iron oxide are introduced into a living body, and alternating current is introduced. A method of locally heating a tumor part using heat generated by hysteresis loss of the ferromagnetic fine particles in a magnetic field, or described in International Publication No. 01/058458 (Special Table 2003-522149) As described above, particles having a dielectric or semiconductive core capable of photothermal conversion and a shell that is conductive are introduced into a living body and irradiated with light capable of photothermal conversion. A method for inducing this has been proposed.

  In recent years, cell transplantation and cell therapy have been actively studied in the field of regenerative medicine, and in order to confirm these transplantation and therapeutic effects at an early stage, it is important to follow the cell dynamics of cell transplantation and cell therapy. ing. Magnetic resonance imaging and the like have been proposed as an effective method for noninvasively monitoring cells transplanted in the living body. Examples of such a method include a method using an inactivated virus envelope labeled with magnetic particles as described in, for example, International Publication No. 05/009478 (Japanese Patent Laid-Open No. 2005-95153). Proposed. On the other hand, in non-invasive living tissue measurement, an optical diagnostic method using near-infrared light having a certain degree of permeability with respect to living tissue has attracted attention and has been studied. In general, light is scattered or absorbed by a living tissue, so that the attenuation rate is high. In order to compensate for the disadvantage, it has been studied to use an agent such as a photocontrast agent. As such a method, as described in, for example, International Publication No. 01/058458 pamphlet (Japanese translations of PCT publication No. 2003-522149), a technique using silica doped with a rare earth light emitter has been proposed.

  Used in the medical field such as the magnetic resonance imaging contrast agent exemplified above, the therapeutic agent used for thermotherapy, the labeling agent used for tracking cell dynamics, or the optical contrast agent used for non-invasive measurement by light Particles made of metal compounds can be surface-modified with biocompatible, tissue-compatible, or cell-compatible substances according to their clinical use, or tissue-specific adsorbents or cell-specific adsorbents, etc. It has been studied to improve the intended function by modifying the surface.

  However, at the present time, when considering clinical application, it cannot be said that sufficient functions are provided.

  Various studies have been conducted on virus particle-like structures encapsulating physiologically active substances. However, there are problems that the physiologically active substance itself is inactivated at the time of producing the structure or is hardly soluble in the solvent at the time of production. . On the other hand, with regard to a preparation encapsulating a non-physiological substance such as a metal or a metal compound, research on drug design and production methods, including effective utilization methods, is not necessarily sufficiently advanced.

  In view of the above situation, the present inventors have advanced development research as an approach that is closest to the practical application to clinical practice by providing particles made of a metal or a metal compound as a structure having a form suitable for medical application. Utilizing biocompatible materials, specific materials that can adsorb and bind to specific materials, materials that can function as labels or reporters, use proteins that provide various possibilities of surface modification, self-assembly The present invention was completed with the belief that a viral protein having the above function was useful for this purpose.

  Accordingly, an object of the present invention is to provide a virus particle-like structure and a production method and application thereof.

  The virus particle-like structure of the present invention contains a virus protein and microparticles containing at least one of a metal and a metal compound (hereinafter also referred to as a metal / metal compound) as a substance to be encapsulated, and substantially It is a virus particle-like structure having a uniform size. That is, the present invention provides a metal or metal compound that is promising for cell-level diagnosis and treatment by incorporating a metal or metal compound effective in diagnostic imaging, tissue regeneration, or thermotherapy into a virus particle-like structure. The present invention provides a virus particle-like structure that encapsulates and a method for optimally forming the structure.

  According to the present invention, a virus particle-like structure containing a virus protein and a metal or a metal compound as an encapsulated substance can be used as an effective preparation for cell diagnosis and treatment with little variation and side effects. In addition, it is possible to provide a virus particle-like structure of uniform size by the stable formation method of this structure.

  The virus particle-like structure of the present invention encloses nanoparticles containing a metal / metal compound as guest molecules and is a substantially uniform size particle, so that the nanoparticle characteristics of the guest molecules can be fully exhibited. .

  In addition, the virus particle-like structure of the present invention is a versatile structure that can be used for various applications by appropriately changing the type of metal or metal compound of the guest molecule according to the purpose. is there.

  The viral protein used in the present invention is not only a carrier for a guest molecule but also cell-directed, and can be modified to have various functions or properties as desired by applying modifications.

  In order to utilize a viral protein having self-assembly and organization ability, the virus particle-like structure of the present invention can be prepared by a simple operation after mixing the components under a predetermined condition.

  Other objects, features, and characteristics of the present invention will become apparent by considering the following description and the accompanying drawings.

FIG. 1 is a transmission electron micrograph showing the in vitro reconstitution of the SV40 virus capsid protein VP1 (pentamer) in the presence and absence of ferrite.

  Upper: When reconstitution was performed with only the VP1 pentamer, a tubular structure was formed at pH 5, and an incomplete structure was formed at pH 7 or 9.

Lower stage: When the VP1 pentamer was reconstituted in the presence of ferrite, formation of small particles having a diameter of 25 to 30 nm was confirmed at pH 5, 7 and 9.
FIG. 2A is a diagram showing the results of Western blotting using anti-VP1 antibody against a hollow virus particle-like structure. 2B is a diagram showing the results of Western blotting using an anti-VP1 antibody against the virus particle-like structure of Example 5, and the lower part of FIG. It is a figure which shows the abundance of Fe ^<2+> . Further, FIG. 2C is a diagram showing the abundance of Fe ^{2+ in} the control of Example 8. FIG. 3 is an electron microscopic observation photograph of molybdenum virus staining of the virus particle-like structure of Example 5 (left: hollow virus particle-like structure, right: virus particle-like structure of the present invention). FIG. 4A is a transmission electron micrograph of the virus particle-like structure of Example 10. FIG. 4B is a diagram showing the results of Western blotting using an anti-VP1 antibody against the virus particle-like structure of Example 10.

  Hereinafter, embodiments of the present invention will be described in detail.

-Virus particle-like structure The structure of the present invention is a virus particle-like structure having a substantially uniform size, containing a virus protein and fine particles containing a metal / metal compound as an encapsulated substance. It is said.

  As used herein, “virus particle-like structure” refers to a structure composed of a plurality of structural elements having a structure similar to a virus particle. Sometimes referred to simply as a “virus-like particle” because of a structure similar to a virus particle or virus particle.

  The “virus particle” is also called a virion, and is a particle structure that satisfies the condition as a virus in terms of morphology, and has a size of 20 to 300 nm. Although some virions have an envelope membrane, the virus particle-like structure of the present invention is typically directed to a structure having no envelope, and is a virus protein and a metal / metal compound that is an encapsulated substance. Containing fine particles. In this specification, “encapsulation” and “encapsulation” are not only those in the enclosed space formed by virus protein aggregates, but also bind to virus proteins (or may be in the form of adsorption, capture, etc.). Also included are those within the viral protein assembly or near the particle surface.

  The virus particle-like structure may further contain a particle formation-promoting factor if necessary or a physiologically active substance if desired. Such physiologically active substances can be bound to viral proteins or encapsulated in viral protein aggregates (eg, low molecular weight compounds such as anticancer drugs, antibiotics, bioactive peptides, polypeptides, proteins, polysaccharides, etc.). Molecular substances) or nucleic acids for gene therapy. The “particle formation promoting factor” will be described later.

-Structure of the present invention The virus particle-like structure of the present invention utilizes the inherent particle-forming ability of the virus, that is, the self-assembly property exhibited by the virus protein. Viral proteins are separated from the nucleic acid and reconstituted in an empty (non-nucleic acid) shell structure reversibly resembling the original viral supramolecular structure in vitro, even if each member is dispersed in pieces. Can be configured. The structure formed by incorporating fine particles containing a metal / metal compound into the assembly during the assembly process of the virus protein is the virus particle-like structure of the present invention. The microparticles containing a metal / metal compound are preferably microparticles that are aggregates of metal / metal compounds, and preferably, a large number of microparticles having a size of nanometer order are incorporated into the viral protein assembly. .

  Since the virus particle-like structure of the present invention is a structure formed on the basis of self-assembly ability and self-organization by a virus protein, the uniformity of the structure is ensured. That is, the structure of the present invention is homogeneous in size, size distribution and shape. The size of the structure may be considered to be a substantially uniform size. “Substantially uniform” means that the size distribution of the structure is within a narrow range. Therefore, it is preferable that the coefficient of variation of the particle diameter is less than 10%, preferably less than 8%, particularly preferably 2 to 5% with respect to the average particle diameter of the structure. If it is in such a range, it means that it is distributed as a monodisperse system. Compared to the particle size distribution of the artificially synthesized particle system, it can be said to be a remarkably uniform system. “Variation coefficient (CV)” is a coefficient represented by 100 × (particle diameter standard deviation / average particle diameter) (%). This is an index indicating that the smaller the numerical value, the more monodisperse particle population. The particle size distribution such as average particle size and coefficient of variation was based on a method by TEM (transmission electron microscope) observation. The shape is the same as or similar to that of the original virus particle, and is spherical or nearly spherical. The size of the virus particle-like structure is preferably 20 to 150 nm, more preferably 20 to 100 nm, and even more preferably 30 to 50 nm because it is a structure without an envelope, and is suitable as a carrier for nanoparticles. It is.

  Among ultrafine particles such as metals, nano-sized particles having a particle diameter smaller than the electron wavelength (about 10 nm) are bulky because the influence of size finiteness on the motion of electrons becomes larger as the quantum size effect. It is known to show unique physical properties different from the body. The virus particle-like structure of the present invention has a special significance as a suitable carrier for taking advantage of such properties of nano-level metals. In order to stably incorporate the metal / metal compound into the viral protein structure as a carrier, a functional group is preferably introduced on the surface of the particle containing the metal / metal compound, and the interaction between the functional group of the particle and the protein is caused. Cause it to occur.

-Metal and metal compound The metal or metal compound included in the virus particle-like structure of the present invention is not particularly limited.

  There is no particular limitation as long as it is a metal or metal compound that is effective for diagnostic imaging, tissue regeneration, or hyperthermia treatment. When encapsulating the virus particle-like structure as an encapsulated substance, when encapsulating the virus particle-like structure with these metals or metal compounds alone or as a composition containing these metals or metal compounds as a main component, The metal or metal compound is preferably in the form of particles.

  Such metals or metal compounds include metals such as iron, manganese, cobalt, nickel, copper, zinc, magnesium, cadmium, molybdenum, barium, titanium, silicon, gadolinium, gold, platinum, vanadium, selenium, aluminum, tin or the like Such composites, oxides, nitrides, carbides or composites of one or more metals as described above, and chelate complexes with organic ligands capable of complexing with the metal ions. Can be mentioned. These metals or metal compounds are preferably those that do not adversely affect living bodies, tissues, and cells according to the application of the present invention.

  Among the above, in particular, when used as an imaging agent, diagnostic agent, or hyperthermia using an alternating magnetic field treatment apparatus in an ultrasonic diagnostic imaging apparatus or a nuclear magnetic resonance imaging diagnostic apparatus, magnetic metal particles or magnetic It is preferable to use metal compound particles. As such a particulate encapsulated substance, iron, iron oxide, or ferrite is more preferable as a main component.

  Such particles mainly composed of iron, iron oxide, and ferrite are magnetic materials such as paramagnetic materials, strong paramagnetic materials, and ferromagnetic materials (hereinafter, these are also collectively referred to as “magnetic materials”). More preferred are paramagnetic substances and strong paramagnetic substances, and it is particularly preferable to use strong paramagnetic substances in terms of no or little residual magnetization.

Specific examples of such a magnetic substance include iron oxides such as triiron tetroxide (Fe ₃ O ₄ ) and γ-heavy sesquioxide (γ-Fe ₂ O ₃ ), various ferrite particles, iron, manganese, cobalt, and chromium. And various alloys such as cobalt, nickel, and manganese, and the ferrite particles are of the MIIO · Fe ₂ O ₃ (MII = Mn, Fe, Co, Ni, Cu, Zn, Mg, Cd) type. Divalent metal salts and the like.

The magnetic particles used in the present invention can be any of magnetite, Fe ₂ O ₃ , Fe ₃ O ₄ , mixed ferrites, other iron-containing compounds including organic ferromagnetic materials, etc., but exhibit the maximum magnetic force. Fe ₃ O ₄ in which ferrite (solid solution of _Fe 3 _{O 4} and _δ-Fe 2 _{O 3)} is a magnetically responsive good, particularly preferred. Ferrite nano-level fine particles with good magnetic properties can be synthesized by controlled precipitation under mild conditions at 4-25 ° C. and near neutral pH as disclosed in JP-A-2002-128523. . The magnetic particles having ferrite as a core can further contain various metal elements such as Zn, Co, and Ni to control magnetic properties.

  Furthermore, as imaging agents and diagnostic agents used for imaging and diagnosis with light, fine particles containing various kinds of probes and metal / metal compounds that can act as light emitters can be suitably used in the present invention. Examples of such metals include molybdenum, copper, barium, zinc, titanium, gadolinium, gold, platinum, cadmium, silicon, vanadium, selenium, tellurium, aluminum, tin, and the like.

  Among these fine particles containing a metal / metal compound, what is called a quantum dot is that the emission wavelength is shifted with respect to the excitation wavelength, the fluorescence lifetime is long, and the wavelength can be controlled by the particle diameter. Therefore, it can be suitably used according to the application. Such quantum dots are prepared as quantum size particles composed of Group II to VI, Group III to V and Group IV elements of the periodic table, and specific compositions include CdS, CdSe, CdTe, ZnS. ZnSe, ZnTe, MgTe, GaAs, GaP, GaSb, GaN, HgS, HgSe, HgTe, InAs, InP, InSb, InN, AlAs, AlP, AlSb, AlS, PbS, PbSe, Ge, Si, alloys thereof, or And mixtures thereof such as ternary and quaternary mixtures. Further, if necessary, the particle having the above-described composition is used as a core, and ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaAs, GaN, GaP, GaAs, GaSb, HgO, You may coat | cover with the shell of the composition which consists of HgS, HgSe, HgTe, InAs, InN, InP, InSb, AlAs, AlN, AlP, AlSb, those alloys, or mixtures thereof. In the case of such a core / shell structure, the band gap energy of the shell is preferably larger than that of the core.

  Further, radioactive rare earth (lanthanide) ionic species known as infrared phosphors can also be used by making them into nano-sized particles (WO 01/058458), and as such rare earths, neodymium, erbium, praseodymium can be used. Etc.

  Furthermore, an acid addition salt of a physiologically acceptable organic acid or inorganic acid can be appropriately selected and used as the metal or metal compound. Examples of such a salt include acetate, bromide, chloride, and citric acid. Salts, maleates, mesylate salts, nitrates, phosphates, sulfates, tartrate salts, oleates, stearates, tosylate salts, calcium salts, meglumine salts, potassium salts and sodium salts.

  Alternatively, examples of the metal or metal compound include complex salts bound to chelating agents. Such a complex salt is a metal atom and a compound serving as a ligand (hereinafter referred to as “ligand component”) or a metal complex in which a ligand is coordinated.

  Here, as the metal salt, iron compounds such as iron chloride, iron bromide and iron iodide; cobalt compounds such as cobalt chloride, cobalt bromide and cobalt iodide; nickel chloride, nickel bromide, nickel iodide and nickel And nickel compounds such as acetylacetonate; palladium compounds such as palladium chloride, palladium bromide and palladium iodide; The ligand component is typically a linear or cyclic polyaminopolycarboxylic acid having an active amino group as a crosslinking chain. Further, triphenylphosphine, 2,2'-bipyridine, 1,5-cyclooctadiene, 1,3-bis (diphenylphosphino) propane and the like can be mentioned. The compound which is the said ligand component can be used individually by 1 type or in combination of 2 or more types.

-Microparticles The metal or metal compound encapsulated as a "guest molecule" in the structure formed by self-assembly of the viral protein of the present invention is mixed with the viral protein in the form of microparticles and taken into the protein structure. . The form, size, and constituent materials of the fine particles are not particularly limited, but it is preferable that a large number of nanometer order fine particles are taken into the virus protein aggregate. The size of such fine particles is not limited, but the average particle size is usually preferably 0.5 to 100 nm, more preferably 1.0 to 50 nm, more preferably 1.5 to 30 nm.

  The fine particles may be particles made of a metal and / or a metal compound. For example, as seen in ferritin, an iron storage protein, the restricted environment of the protein shell may promote crystal nucleation and aggregation of metal particles.

  Alternatively, the fine particles may be composite fine particles including a metal and / or a metal compound and a compound having a binder function for binding them. As such a binder compound, a water-insoluble organic polymer is desirable. Examples of monomers for obtaining such organic polymers include aromatic vinyl compounds, α, β-unsaturated carboxylic acid esters, α, β-unsaturated carboxylic acid amides, α, β-unsaturated nitrile compounds. , Halogenated vinyl compounds, conjugated diene compounds, lower fatty acid vinyl esters, and the like can be used.

  It is desirable that the surface of the fine particle containing a metal or a metal compound that is an encapsulated substance has a positive or negative charge as a surface charge. By having a negative surface charge on the surface of the fine particles, the metal / metal compound interaction of the encapsulated substance occurs, and the inclusion is increased and stabilized. Such surface charges also contribute to the stabilization of the fine particles that are encapsulated by interacting with the side chains of the amino acid residues of the viral protein.

  The negative charge may be such that a carboxyl group, a phosphoric acid group, or a sulfonic acid group exits on the surface of the fine particles. For example, there is a method of coating fine particles containing a metal / metal compound with a carboxylic acid / carboxylate. Suitable acids for introducing a negative charge include, for example, lactic acid, citric acid, tartaric acid, succinic acid, dimercaptosuccinic acid, maleic acid, fumaric acid and ascorbic acid. Examples of positive charges include amino groups and guanidyl groups.

  In order to expose the charged functional group on the surface of the particle body, a charged group may be introduced into the organic polymer.

  It is also desirable to surface treat the organic polymer for the binder function of the metal / metal compound. As the surface treatment functional group, general surface treatment groups such as amino group, carboxyl group, thiol group, aldehyde group, and carbodiimide group can be used, but are not limited thereto.

  The metal / metal compound may be the same type of metal or metal compound, or may be a combination or mixture of different types of metals or metal compounds.

  As another preferred embodiment of the fine particles containing a metal / metal compound, nanoparticles having a core / shell structure are also exemplified. The fine particles with such a structure can be coated with a material different from the particle core and functionalized without changing the size and shape of the core particles, or exhibit different characteristics from both the core and shell bulk materials. Can be expected. For example, when the surface of the light-emitting nanoparticle is exposed, a large number of defects existing on the surface of the nanoparticle become a light emission killer, resulting in a decrease in light emission efficiency. Therefore, a core / shell structure is formed by coating with a shell material having a band gap larger than the band gap corresponding to the emission wavelength of the nanoparticles.

  Examples are nanoparticles composed of metals or metal-like materials in which the core is made of a non-conductive material, for example a dielectric material or a semiconductor material, and the shell layer is conductive (WO 01/058458). The nature of such materials affects the properties of the particles. As an example, a mode in which a silica core doped with a rare earth light emitter such as neodymium, erbium, or praseodymium is used as silica nanoparticles can be shown. Dielectric materials include silicon dioxide, titanium dioxide, polymethylmethacrylate (PMMA), polystyrene, gold sulfide, and macromolecules such as dendrimers. Examples of semiconductor materials include CdSe, CdS, and GaAs.

It is preferable that 0.002-50 weight part of microparticles | fine-particles are contained with respect to 100 weight part of viral proteins, It is more preferable that 0.0025-50 weight part is contained, It is further containing 0.1-50 weight part Preferably, 4 to 30 parts by weight are contained. Although the range may vary depending on the size and density of the fine particles, it is generally preferable that 3 × 10 ⁸ to 4 × 10 ¹¹ particles are contained per 1 μg of virus protein. More preferably, ¹⁰ to 4 × 10 ¹¹ particles are contained.

-Virus protein type The structure of the present invention makes use of the homogeneity of the structure of the virus particles, the self-assembly ability and self-assembly ability of the virus protein to create a cavity (so-called "cage" structure) within the protein assembly supramolecule. It is a new nanostructured structure that allows the functions of incorporated metal / metal compounds to be demonstrated at the nano level.

  There are various types of viral proteins, but the structure of the present invention is not particularly limited. A capsid which is a protein outer shell is preferable. Capsids are composed of unit particles called capsomers, and it is known that capsomer proteins exist as single or plural proteins. Any capsid protein may be used in the present invention. Of these, VP1 or VP2 capsid protein is preferable.

  In addition, examples of viral proteins used include proteins bound to viral nucleic acids (DNA or RNA), proteins enclosing the protein-nucleic acid complex, and proteins that line the capsid. Furthermore, in a virus having an envelope, any of the envelope proteins that bind to the lipid membrane constituting the envelope or together form the membrane can be used as the viral protein of the structure of the present invention. Moreover, as long as the particle-forming ability is maintained and exhibited, a plurality of proteins derived from different viruses or different types of virus proteins of the same virus may be used in appropriate combination. Specifically, two types of capsid proteins, such as VP1 and VP2, may be used together.

-Virus It does not specifically limit to the kind of virus which supplies viral protein, The various protein from various viruses can be used for the structure of this invention. As viruses to which the present invention can be applied, specifically viruses of the genus Papovavirus such as SV40 virus, JCV virus, BKV virus; hepadnavirus (such as hepatitis B virus), adenovirus, flavivirus (such as Japanese encephalitis virus), Herpesvirus (herpes simplex virus, varicella-zoster virus, cytomegalovirus, EB virus, etc.), pox virus, parvovirus (adeno-related virus, etc.), orthomyxovirus (influenza virus, etc.), rhabdo virus (rabies virus, etc.) , Retroviruses (acquired immune deficiency syndrome virus, etc.), hepatitis C virus, lentivirus, herpes virus, bacteriophage, influenza virus, Sendai virus, vaccinia virus Others can be mentioned viruses such as baculovirus. Of these, viruses exhibiting an icosahedral structure are preferred.

  Since the affinity and adsorption specificity for cells of the host (including human subjects who receive administration of the structure of the present invention) differ depending on the virus, considering the directivity of the necessary structure, It is desirable to select the type and use the protein. Thereby, specific targeting performance can be provided.

  When a viral protein is used in a preparation applied to a living body, it is its safety that immediately becomes a problem. Some viruses have pathogenicity, some structural proteins have cytotoxicity, and others have antigenicity. From this point of view, it can be said that virus proteins from the papovavirus genus, particularly SV40 virus, JCV virus and BKV virus, are preferable. These viruses are considered to be harmless to humans or extremely low in pathogenicity, with almost no pathological signs resulting from infection of the virus. The papovavirus that infects primates, among them SV40 virus, which has high safety to humans and high structural stability, has the advantage that no antigenicity is observed even when administered to humans. On the other hand, since the JCV virus is a virus that infects brain regions, its protein has the property of passing through the blood-brain barrier, but has antigenicity. In the structure of the present invention, a virus protein derived from the genus Papovavirus is particularly preferable.

-Virus protein mutants or variants Virus proteins may be obtained by purifying native proteins obtained from natural viruses, but virus proteins mutants or variants with the necessary modifications are also preferably used. can do. Alternatively, the native viral protein and its mutant or variant may be used together. The reason for using such a mutant or variant is that the antigenicity of the virus particle-like structure is reduced or it is difficult to be recognized by the immune surveillance mechanism (the so-called “stealth” state). I can expect. Alternatively, from the viewpoint of providing a targeting function, there is a need to enhance affinity for specific organs, tissues, and cells. There is also a need to increase the stability of the structure. Furthermore, there is a need to meet the demand for forming a portion that can add various functions to the protein surface. Thus, it is possible to respond to the demands from various viewpoints by appropriate modification of the protein molecule.

  By enhancing the hydrophilic tendency of the viral protein, the blood stability can be increased and the concentration in the blood can be maintained for a long time. In addition, in order to improve the blood retention, it is possible to increase the hydrophilicity by introducing polyethylene glycol (PEG) or polyalkylene oxide chain on the protein surface. By appropriately changing the length of the oxyethylene unit and the ratio of introduction, the function can be adjusted. As PEG, polyethylene glycol having 10 to 3500 oxyethylene units is preferable.

  A “functional substance” may be added as a functional functional group to the end of the polyethylene glycol or polyalkylene oxide chain introduced on the surface of the viral protein. In addition to the effect of introducing a polyalkylene oxide chain, a structure in which a polyalkylene oxide chain having a “functional substance” bonded to the tip is immobilized has a function substance that is not obstructed by the polyalkylene oxide chain. ", For example, functions such as specific organ directivity and specific tissue directivity are sufficiently exerted as a" recognition element ".

  Viral particle-like surfaces, in fact viral protein surfaces, as drug molecules, reporter labels (eg chromophores, enzymes or radioactive labels) or affinity ligands (eg antibodies or antibody fragments, specific A partner fragment that specifically binds (specifically, biotin or streptavidin), an oligopeptide, an oligonucleotide, or an oligosaccharide) may be functionalized by coupling to a viral protein that constitutes the particle. Direct coupling is bound by a functional group of the protein, such as an amino group, an oxycarbonylimidazole group, an N-hydroxysuccinimide group, which is highly reactive.

  Affinity ligands include monoclonal antibodies, polyclonal antibodies, antibody fragments, nucleic acids, oligonucleotides, proteins, oligopeptides, polysaccharides, saccharides, nucleic acid molecules encoding peptides, lectins, cell adhesion factors, antigens, drugs and other ligands Etc.

-Production of Mutant or Variant As described above, the viral protein may be a mutant or variant that is functionally equivalent to the native viral protein. That is, it may be a variant or a variant that retains the ability to form particles and that has modified the target tissue or cell directivity of the native viral protein. The mutant or variant in this case can be obtained by mutagenesis, chemical modification or gene recombination techniques. A virus protein mutant or variant is one or more, preferably 1 or several, more preferably 1 to 24, and even more preferably 1 to 15 amino acid residues in the amino acid sequence constituting the viral protein. A protein consisting of an amino acid sequence in which a group is deleted, added or substituted by another amino acid residue. Further, such a mutant or modified substance has a particle forming ability.

  Here, “deletion” means that one or more amino acid residues are lost from the polypeptide chain of the native protein. Conversely, “addition” means insertion of one or more amino acids into a polypeptide chain, and it may be put in one form in the form of a peptide. “Substitution” refers to replacement of an amino acid residue at a specific site with another type of amino acid.

  The term “functionally equivalent” is an amino acid sequence modified by deletion, substitution, addition and / or insertion of one or more amino acids in the amino acid sequence, or in the amino acid sequence, eg phosphorylated Alternatively, it is a sequence in which the side chain of an amino acid residue is chemically modified by dephosphorylation, glucosylation or deglucosylation, etc., but nevertheless, substantially the same action or activity as that of a natural viral protein is maintained. It corresponds to that. Such a functionally equivalent variant may occur as a natural biological mutation, but may be produced by a method of mutagenesis at a specific site or mutagenesis at an unspecified site. Furthermore, a variant can be manufactured using well-known techniques, such as a chemical modification method, enzymatic cleavage, and / or a ligation reaction. Appropriate nucleic acid molecules operatively linked to expression control sequences are expressed in host cells, or by recombinant means of expressing recombinant DNA cloning vectors containing such recombinant DNA molecules, or chemically or biologically. It can also be prepared by chemical synthesis (extracellular).

  A virus protein mutant or native protein artificially synthesized partly or entirely, or a variant obtained by modifying a protein of biological origin has a function not found in the virus-derived protein as described above. Or having inappropriate properties such as improved antigenicity.

  Viral protein variants or variants may have lost particle-forming ability or may have enhanced antigenicity. Such a mutant or modification cannot be used in the structure of the present invention. Therefore, there is a need to check for variants or variants of viral proteins in advance to see if they exhibit such disadvantageous properties.

  The virus protein, the virus protein mutant or variant is desirably at least one of a VP capsid protein such as SV40 virus, JCV virus or BKV virus and a variant or variant thereof. This is because, among the viral proteins, these capsid proteins have known amino acid sequences, can be easily obtained, and can be purified.

  In the case of the SV40 VP1 capsid protein, JCV capsid protein, and BKV capsid protein, the present inventors have found that there is an exogenous peptide insertion site capable of retaining particle-forming ability and cell adhesion. It was. It was also confirmed that the cell directivity was changed depending on the type of foreign peptide chain inserted. From these findings, it can be seen that viral proteins have variability that selectively and specifically changes their carrier functions.

-Particle formation promoting factor The virus particle-like structure of the present invention may further contain a particle formation promoting factor that promotes self-assembly and self-assembly of the viral protein and the fine particles. Even when virus protein and metal (compound) -containing particles are mixed in the presence of such a particle formation promoting factor, the self-assembled organization of the virus protein is promoted, and the structural elements are efficiently As a result of the assembly, the organization progresses, and as a result, structures of almost the same size are formed.

  Examples of the particle formation promoting factor include capsid protein of virus particles, DNA, RNA, biopolymer, artificial polymer, compound monomer, or ferrite particles whose surface is coated with these. Examples of the biopolymer include DNA, RNA, or protein. Examples of the artificial polymer include polyethyleneimine, poly-L-glutamic acid, poly-L-lysine, poly-L-aspartic acid, poly-L-arginine and the like. Examples of the compound monomer include citric acid and dimercaptosuccinic acid. In the viral capsid protein, the region having particle formation promoting activity is known to be an amino-terminal region. For example, in the SV40 virus capsid protein VP2, a polypeptide having an amino acid sequence of at least positions 1 to 272 can function as a particle formation promoting factor. The capsid protein VP1 forms a pentamer, but its structure changes in the presence of a particle-forming factor and associates with each other to form a regular icosahedron structure. In the case of ferrite particles coated with a biopolymer or the like, the surface preferably has a charge (for example, a carboxyl group).

Production Method The above virus particle-like structure can be produced as follows.

  In the method for forming a virus particle-like structure of the present invention, a viral protein and / or a mutant or a variant thereof and a microparticle containing at least one of a metal and a metal compound as an encapsulated substance are monovalent to 100 mM to 500 mM. It is characterized by incubation at 15 to 30 ° C. at pH 5 to pH 10 in the presence of a cation and a divalent cation of 2 μM to 50 mM.

  Specifically, the virus protein purified using the existing protein purification technology is mixed with the fine particles containing the metal or metal compound as the encapsulated substance with sufficient stirring (I) and step (I And (II) in the presence of 100 mM to 500 mM monovalent cation and 2 μM to 50 mM divalent cation at pH 5 to pH 10 at 15 to 30 ° C.

After step (I), before step (II), a salt (eg, NaCl, CaCl ₂ etc.), a chelating agent (eg EDTA, EGTA etc.), and if necessary a reducing agent (eg DTT) etc. The containing buffer may be added and the mixed solution may then be incubated at 4 ° C. for a predetermined time. In one preferred embodiment of step (II), the solution is allowed to react for 15-20 hours, preferably 16 hours, at 15-30 ° C., 100 mM to 500 mM monovalent cation and 2 μM to 50 mM divalent cation at pH 5, 7 or pH 9. Dialyze with a solution containing a valent cation.

  Examples of the monovalent cation include sodium ion, potassium ion, ammonium ion, and the like, preferably sodium ion. Examples of the divalent cation include magnesium ion and calcium ion, and calcium ion is preferable. Furthermore, the concentration of the monovalent cation is desirably 100 to 500 mM, more desirably 100 to 200 mM, still more desirably 140 to 160 mM, and most desirably 150 mM. Further, the concentration of the divalent cation is preferably 1 to 5 mM, and more preferably 2 mM.

  In the production of a virus particle-like structure having a positive or negative charge as a surface charge and containing a microparticle containing a metal / metal compound and a virus protein and / or a variant or variant thereof, the virus protein, Prepared by mixing 0.01 to 100 parts by weight of fine particles containing metal / metal compound with 360 parts by weight of virus protein and dialyzing against an aqueous solution containing monovalent metal salt and divalent metal salt. The manufacturing method characterized by the above is also the method of the present invention.

  In the production method, the weight ratio of fine particles containing a metal / metal compound and having a negative charge to the viral protein is preferably 0.2 or more, more preferably 0.2 to 3, and More preferably, it is 2 to 0.27.

  Furthermore, in the production method, examples of the monovalent metal salt include sodium salts, potassium salts, ammonium salts, and the like, and sodium salts are preferable. The divalent metal salts include magnesium salts, calcium salts, and the like. Examples of the salt include calcium salts.

  Fine particles containing a metal / metal compound can be produced by applying a technique already reported. Clusters of cohesive metals, metal ions or metal compounds are formed as nanoparticles. Alternatively, it is deposited on a binder or linked via a linker molecule. Either by solution metal reduction or by a colloid-based deposition process.

The metal / metal compound may be coated with a metal-free material such as an organic substance, or conversely, particles made of a non-metallic compound may be coated with a metal or a metal compound. Furthermore, for example, Au 2 _S reduction of the particle on the Au, such as particles having a Au 2 _S core coated with gold by (reduction of chloroauric acid by sodium sulfide), a core / shell structure in which the metal is also composed of only laid metal compound The particles can also be made. As another method, a metal or a metal compound may be mixed with a monomer, polymerized, and dispersed in the polymer.

Aspects of Use When the virus particle-like structure of the present invention is introduced into a living body, the metal / metal compound encapsulated in the virus protein aggregate exhibits a predetermined function. In the case of a magnetic metal, heat due to hysteresis loss is dissipated. In the case of magnetic resonance, the signal is emitted. In the case of a light emitter, light is emitted, and information in the living body is also provided. In the case of fever, biological information added to the signal can be used for treatment and diagnosis. Therefore, the preparation containing the virus-like structure is for achieving such an object and is included in the present invention.

-Imaging agent Specifically, the said formulation is an imaging agent, the diagnostic agent with respect to a tumor, or a therapeutic agent. More specifically, the preparation is desirably a contrast agent for an ultrasonic diagnostic imaging apparatus, a nuclear magnetic resonance imaging diagnostic apparatus, or an X-ray diagnostic imaging apparatus as an imaging agent.

Various paramagnetic metals can be used as the metal atom of the paramagnetic metal compound used in the magnetic resonance contrast agent. Magnetic particles that are encapsulated in the virus particle-like structure of the present invention and are suitably used as a magnetic resonance contrast agent are those containing iron, iron oxide, and ferrite as main components. Magnetite, Fe ₂ O ₃ , Fe ₃ O ₄ , mixed ferrites, other iron-containing compounds including organic ferromagnetic materials, etc. can be used, but ferrite (Fe ₃ O ₄ that exhibits the maximum magnetic force) ₃ O ₄ and δ-Fe ₂ O ₃ (solid solution) are particularly preferable because of their good magnetic response.

  As a metal atom of a paramagnetic metal compound used for a magnetic resonance contrast agent, in addition to the above iron, a lanthanoid element having an atomic number of 57 to 70, particularly gadolinium (Gd), dysprosium (Dy), ytterbium (Yb), praseodymium ( Pr), neodymium (Nd), samarium (Sm), terbium (Tb), holmium (Ho), erbium (Er), chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni), copper ( And Cu) transition metal species.

  When the transition metal is used, it may be an embodiment in which a complex formed by reacting a divalent or trivalent ion with a chelating compound is included in a viral protein assembly. The reason is that even if the protein is decomposed and paramagnetic metal ions are released from the structure, the toxicity is reduced in the form of a complex. For example, free gadolinium is deposited in tissues and shows strong toxicity to the liver and bone marrow, but is rapidly excreted from the kidney by chelation. Thus, the chelating compound reduces the toxicity of the paramagnetic metal, which is a heavy metal, and has usefulness in the function of assisting its administration, in vivo transfer, excretion, and the action of the paramagnetic metal with protons. Demonstrate.

  The chelating compound is not particularly limited as long as it can form a complex with a paramagnetic metal atom and has a suitable affinity for the virus particle-like structure of the present invention. Specifically, a linear or cyclic polyaminopolycarboxylic acid having an active amino group as a crosslinking chain and having a bifunctional structure capable of capturing a metal ion to form a complex is preferable. For example, a derivative of DTPA (diethylenetriaminepentaacetic acid) and a salt thereof are conceivable. Specific examples include monoalkylamide DTPA, dialkylamide DTPA, monoarylamide DTPA, diarylamide DTPA, monoalkyl ester DTPA, dialkyl ester DTPA, monoaryl ester DTPA, diaryl ester DTPA, alkylated DTPA, and the like. Among these compounds, alkyl includes 120 alkyl groups, and aryl includes phenyl and naphthyl. The aryl may be substituted with an alkyl, a halogen atom or the like.

  Other examples of chelating compounds include TTHA (triethylenetetraaminehexaacetic acid), EDTA (ethylenediaminetetraacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra Acetic acid), EHPG (N, N-ethylenebis [2- (2-hydroxyphenyl) glycine]), Cyclam (1,4,8,11-tetraazacyclotetradecane), NTA, HEDTA, BOPTA, NOTA, DO3A, Examples include HPDO3A, EOB-DTPA, TETA, HAM, DPDP, porphyrin and derivatives thereof (EOB means ethoxybenzyl).

  The paramagnetic metal atom ion and the chelating agent are chelated by a conventional method. As a result, the following compounds are exemplified as examples of the produced paramagnetic metal or derivatives thereof: Gd-DTPA, Gd-EOB-DTPA, Yb-EOB-DTPA, Dy-EOB-DTPA, Mn-DTPA , Gd-BOPTA, Gd-DOTA, Gd-HPDO3A. Also suitable are compounds based on metal-containing macrocycles such as gadobutrol.

・ Therapeutic agents Hyperthermia for cancer belongs to the category of noninvasive cancer therapies, such as laser therapy and photodynamic therapy. Thus, although thermotherapy for cancer is a treatment method having unique characteristics and excellent aspects, it has not always been actively adopted in the cancer treatment field. Hyperthermia is not used alone, but rather is used in combination with radiation and anticancer agents for the purpose of enhancing the effects of radiation and anticancer agents. There are several reasons for this, but it can be said that there was no reason to replace the conventional method because the therapeutic effect could not necessarily achieve results superior to other therapies. For this reason, it is considered that there is still room for improvement in members, drugs, and devices used for cancer thermotherapy.

  At present, the target of this therapy is locally advanced cancer or recurrent cancer that is difficult to cure with conventional therapy. There are two types of hyperthermia: a method that warms the whole body (whole body thermotherapy) and a method that warms the cancer and its vicinity (local thermotherapy). In general, local hyperthermia is mainly performed, and the local area is heated by a device using microwaves, electromagnetic waves (alternating magnetic field), and ultrasonic waves. Heating from the outside of the body is the most common method, but there are other methods such as placing the device in the lumen of the esophagus, rectum, uterus, bile duct, and several electrode needles in the cancer tissue. Attempts have also been made to add and heat. It is known that the effect on cancer is obtained at 41 ° C. or higher, but becomes particularly strong at 42.5 ° C. or higher. Cancers close to the surface of the body can be heated relatively easily to the target temperature, but cancers deep in the body are often difficult to warm up due to obstruction of fat, air, and bones. Often the effect of therapy is inadequate. Therefore, if the preparation of the present invention in which magnetic particles are encapsulated in a virus particle-like structure is introduced into the body and used as a heating element for thermotherapy, cancer tissue in the back of the body, scattered cancer cells, cells of minute initial cancer Effective treatment is possible.

  When the preparation of the present invention is used as such a thermotherapy agent, for example, in a subject's vein, a magnetic particle comprising a metal or a metal compound capable of expressing magnetism such as ferrite (the magnetic particle-containing preparation of the present invention) And within a period of 1 minute to 48 hours, preferably 30 minutes to 36 hours after the start of the administration, the subject is exposed to energy irradiation as described below. By doing so, the temperature of the tumor tissue adjacent to the magnetic particles can be raised, and treatment can be performed with little effect on normal cells. On the other hand, the magnetic particle-containing preparation may be injected directly in the vicinity of the tumor tissue of the subject, and 0.5 to 36 hours, preferably 10 to 24 hours after the start of injection, The temperature of the tumor tissue adjacent to the encapsulated magnetic particles can be increased by irradiating the subject with cancer cells with energy irradiation described later.

  The energy irradiation preferably includes alternating magnetic field irradiation or ultrasonic irradiation, and the alternating magnetic field irradiation is preferably performed at a frequency of 25 to 500 kHz.

-Composition for cell introduction Further, the present invention also includes a composition for introducing microparticles containing the above-mentioned virus particle-like structure and containing the metal / metal compound into cells.

  When a target metal / metal compound or a particle containing the target metal is introduced into the target cell membrane, it can be easily and efficiently transferred according to the form in which the introduced metal is enclosed in a virus particle-like structure. Can do. The composition of the present invention contains such a virus particle-like structure and further contains necessary additional substances. Examples of the additive substance include a substance for stabilizing the structure, salts, chelating agent, polyethylene glycol, lysolecithin, glycerol oleate as a fusion accelerator, and the like. Suitable techniques for transfection are well known and include electroporation, microinjection, lipofection, virus transfection and protoplast fusion.

  The metal / metal compound molecules carried in the virus particles and introduced / transferred into the cells are made into a particle form as described above, and are taken into the particle-like structure formed by the virus protein. For example, it is incorporated into the protein coat of the virus coat or capsid. When used generally for gene therapy or other in vivo applications, the virus is usually disabled for safety reasons. As a result, these viruses can infect host cells but cannot replicate or assemble new virions. In addition, new cells cannot be infected, that is, replication cannot be made incompetent. Adenoviruses used for gene therapy are also preferred viral systems for use in the present invention. The above virus particle-like structure is also disabled.

[Example]
The following examples are only preferred embodiments within the scope of the present invention. In addition, the apparatus name used in the examples, the concentration of the materials used, the amount used, the processing time, the processing conditions such as the processing temperature, the processing method, etc. are merely preferred examples within the scope of the present invention.

(Example 1)
Synthesis of ferrite particles Synthesis was performed by a general coprecipitation method. Specifically, 25 mL of an aqueous solution obtained by mixing 8.3 mL of 0.1 M FeCl ₂ aqueous solution and 16.7 mL of 0.1 M FeCl ₃ aqueous solution was added to 50 mL of 200 mM NaOH aqueous solution, and stirred for 1 hour at room temperature to obtain ferrite particles ( A solid solution of Fe ₃ O ₄ and γ-Fe ₂ O ₃ was prepared.

  The ferrite particles prepared here were confirmed to have an average particle diameter of 8 nm by observation with a transmission electron microscope.

(Example 2)
Synthesis of Citric Acid-Immobilized Ferrite Particles 50 mL of 100 mM sodium citrate aqueous solution (pH 7.0) is added to 180 mg of the ferrite particles prepared in Example 1, and citrate molecules are applied to the ferrite particle surface while performing ultrasonic treatment. Was fixed. The obtained citric acid-immobilized ferrite particles were dispersed in ultrapure water by dialysis. Thereafter, centrifugation was performed to remove aggregates contained in the dispersion, and the supernatant was collected as citrate-immobilized ferrite particles. The obtained citric acid-immobilized ferrite particles were confirmed to be dispersed with an average particle diameter of about 12 nm in an aqueous solution by a dynamic light scattering method.

(Example 3)
Synthesis of dimercaptosuccinic acid-fixed ferrite particles 50 mL of 100 mM sodium dimercaptosuccinate aqueous solution (pH 7.0) was added to 180 mg of the ferrite particles prepared in Example 1, and the surface of the ferrite particles was subjected to ultrasonic treatment. Hedimercaptosuccinic acid molecules were immobilized. The obtained dimercaptosuccinic acid-immobilized ferrite particles were dispersed in ultrapure water by dialysis. Thereafter, centrifugation was performed to remove aggregates contained in the dispersion, and the supernatant was recovered as dimercaptosuccinic acid-immobilized ferrite particles. The obtained dimercaptosuccinic acid-immobilized ferrite particles were confirmed to be dispersed with an average particle diameter of about 12 nm in an aqueous solution by a dynamic light scattering method.

(Example 4)
Reconstitution of Ferrite-Containing Virus-Like Particles The purified SV40 VP1 pentamer protein was mixed with 10 μg of the citric acid-immobilized ferrite particles of Example 2 and 3.6 × 10 ¹² particles (4800 ng). The total volume of this mixed solution was adjusted to 100 μL with 20 mM Tris-HCl (pH 7.9), 150 mM NaCl, 5 mM EGTA, 5 mM DTT solution. The mixed solution was adjusted to pH 5 (HCl [pH 5.0]), pH 7 (20 mM Tris-HCl [pH 7.0]), and pH 9 (20 mM Tris-HCl [pH 9.0]) containing 150 mM NaCl and 2 mM CaCl _2. The solvent was replaced by dialyzing the mixture at room temperature (22 ° C.) for 16 hours for each adjusted solution.

  This solution was collected, and the assembly mode of the VP1 pentamer in the substituted solvent was observed using a transmission electron microscope. The addition of ferrite particles confirmed the formation of a T = 1 virus particle-like structure, which is an aggregate of 12 VP1 pentameric proteins.

  Such reconstitution experiments on ferrite-containing virus-like particles have shown that nanomagnetic ferrite particles can be incorporated into virus-like particles.

(Example 5)
Formation efficiency of virus particle-like structure when the amount of ferrite was changed Purified SV40 VP1 pentamer protein, 10 μg of citric acid-immobilized ferrite particles of Example 2, 0.3 × 10 ¹⁰ particles (48 ng), 3 .6 × 10 ¹¹ particles (480 ng), 1.1 × 10 ¹² particles (1500 ng) and 3.6 × 10 ¹² particles (4800 ng) were mixed. This mixture was adjusted to a total volume of 100 μL with 20 mM Tris-HCl (pH 7.9), 150 mM NaCl, 5 mM EGTA, 5 mM DTT solution. A solution containing 150 mM NaCl and 2 mM CaCl ₂ was mixed with this mixed solution at pH 5 (20 mM Tris-HCl [pH 5.0]), pH 7 (20 mM Tris-HCl [pH 7.0]) and pH 9 (20 mM Tris-HCl [pH 9.0]. ]) Was replaced by dialyzing for 16 hours at room temperature (22 ° C.).

  This solution was collected, and the assembly mode of the VP1 pentamer in the substituted solvent was observed using a transmission electron microscope. Usually, when dialyzed only with the VP1 protein, a tubular structure was formed. Under these conditions, depending on the amount of ferrite particles added, formation of a T = 1 virus particle-like structure, which is an aggregate of 12 VP1 pentameric proteins, was confirmed.

As shown in FIG. 1, formation of a T = 1 virus particle-like structure, which is an aggregate of 12 VP1 pentameric proteins, was confirmed by the addition of ferrite particles. In addition, what was shown in FIG. 1 added the ferrite particle 3.6 * 10 < ¹² > particle | grains (4800ng).

(Example 6)
Imaging Agent Rat adrenal pheochromocytoma cell line PC-12 was cultured according to a conventional method, 2 × 10 ⁵ cells per 2 mL of medium were seeded on a 35 mm diameter plate (6-well plate), cultured overnight, and then 35 μL of a virus particle-like structure suspension containing 3.6 × 10 ¹² particles (4800 ng) of ferrite particles prepared in Example 5 of Example 5 was added to PC-12 cells (2 mL culture), and Centrifuge at 1,600 × g for 10 minutes. After centrifugation, the medium was changed, and the plate was incubated at 37 ° C. under 5% CO ₂ for 20 hours to introduce a virus particle-like structure including ferrite particles into PC-12. In addition, it produced by the method similar to the above using the virus particle-like structure which does not include a ferrite particle as negative control.

Next, the above-described virus particle-like structure-introduced PC-12 cells and the virus particle-like structure that does not contain ferrite particles are peeled off from the plate by trypsin treatment and suspended in 0.2 mL of medium (about 1 × 10 ⁶ cells / mL), 0.2 mL of the cell suspension was collected in an NMR sample tube, and magnetic resonance measurement (CSI for 2 Tesla animal experiments: Bruker, Karlsruhe, Germany) was performed. The spin echo method was used as the imaging method, and the region of interest (FOV) was set to 35 mm × 35 mm, the slice thickness was 4 mm, and the matrix was 256 × 128, and the T ₁ -weighted image and the T ₂ -weighted image were observed. The imaging conditions for the T ₁ -enhanced image required a measurement time of about 2 minutes with a repetition time (TR) of 500 ms, an echo time (TE) of 18 ms, a flip angle of 90 °, and two scans. Moreover, the imaging conditions for the T ₂ -weighted image required a measurement time of about 17 minutes with a repetition time of 2000 ms, an echo time of 80 to 120 ms, a flip angle of 90 °, and four scans. Each original data was imaged by adding zero fill, and finally image data of a matrix 256 × 256 was obtained. As a result, when the signal intensity of the T2-weighted image of the PC-12 cell suspension into which the virus particle-like structure not including ferrite particles was introduced was set to 100, the virus particle-like structure including ferrite particles was introduced. The signal intensity of the T2-weighted image of the PC-12 cell suspension was 40.

  From the above, the virus particle-like structure including the ferrite particles of the present invention is expected to function effectively as an imaging agent or a diagnostic agent for a living body.

(Example 7)
Thermotherapy agent Cell line PC-12 derived from rat adrenal pheochromocytoma was cultured according to a conventional method, 2 × 10 ⁵ cells per 2 mL of medium were seeded on a 35 mm diameter plate (6-well plate), cultured overnight, and then the above-mentioned 35 μL of a virus particle-like structure suspension containing 3.6 × 10 ¹² particles (4800 ng) of ferrite particles prepared in Example 5 of Example 5 was added to PC-12 cells (2 mL culture), and Centrifuge at 1,600 × g for 10 minutes. After centrifugation, the medium was changed, and the plate was incubated at 37 ° C. under 5% CO ₂ for 20 hours to introduce a virus particle-like structure including ferrite particles into PC-12. In addition, it produced by the method similar to the above using the virus particle-like structure which does not include a ferrite particle as negative control.

  Next, an optical fiber is used to measure the temperature of the PC-12 cells in the plate using the PC-12 cells into which the virus particle-like structure is introduced and a virus particle-like structure plate that does not include ferrite particles. When a thermometer was placed and irradiated with an alternating magnetic field for 2 minutes under the conditions of a resonance frequency of 300 KHz, a coil diameter for generating a magnetic field of 200 mm, a magnetic field strength of 6 mT, a current of 225 A, an output of 3 kW, and a distance of 20 mm from the applicator, virus particles The temperature of the PC-12 cell plate into which the like structure was introduced was higher by about 6 ° C.

  From the above, the virus particle-like structure including the ferrite particles of the present invention is expected to function effectively as a thermotherapy agent for living bodies.

(Example 8) Analysis of ferrite-containing virus-like particles by sucrose density gradient centrifugation Density of 20% (w / v) to 40% (w / v) sucrose (20 mM Tris-HCl (pH 7.9)) 20 μl of a virus-like particle solution containing 3.6 × 10 ¹² particles (4800 ng) of the ferrite particles prepared in Example 5 described above was added to 600 μl of the gradient solution, and centrifuged at 50,000 rpm for 1 hour. After centrifugation, it was fractionated into 12 fractions by sucking 55 μl from the top.

  Of these, 7 μl was subjected to Western blotting using an anti-VP1 antibody to examine the fraction containing VP1. As a control, the same experiment was conducted with a hollow virus-like particle solution.

  Hollow T = 1 virus-like particles were detected in the 3-4 fraction and large virus-like particles were detected in the 7-9 fraction (FIG. 2-A), whereas ferrite-containing T = 1 virus-like particles Was detected in the 7-9 fraction (FIG. 2-B top).

Also, with the remaining 48 μl, add 50 μl of 6M HCl, 100 μl of 908 mM ascorbic acid and let stand for 10 minutes, then leave 150 μl of 1.04 mM 5-Br-PSAA and 150 μl of 3.66 M sodium acetate, and let stand for 20 minutes. The fraction containing Fe ²⁺ derived from ferrite was examined by measuring the absorbance at 558 nm. As a control, the same experiment was performed for the ferrite solution.

  Ferrite itself was detected in the 12 fraction (bottom of FIG. 2-C), whereas ferrite-containing virus-like particles were detected in the 7-9 fraction (FIG. 2-B).

  From the above, it can be seen that the ferrite-containing virus-like particles have a higher density than the hollow virus-like particles and the ferrite is adsorbed.

(Example 9) Electron microscope observation of ferrite-containing virus-like particles by ammonium molybdate staining A virus-like particle solution containing 3.6 × 10 ¹² particles (4800 ng) of ferrite particles prepared in Example 5 described above was added to ammonium molybdate. After dyeing, the sample was observed using a transmission electron microscope. As a control, a similar experiment was performed on a solution in which ferrite was added to hollow virus-like particles.

  While ferrite could be observed in the center of all ferrite-containing virus-like particles (Fig. 3 left), the control was able to observe the state where ferrite was attached around the virus-like particles, but ferrite was observed in the center. It was not possible (Fig. 3 right).

  From the above, it can be seen that all of the ferrite-containing virus-like particles contain ferrite.

(Example 10) Effect of assembly mode of VP1 pentamer protein by ferrite surface coating material Purified SV40 VP1 pentamer protein, 10 μg of dimercaptosuccinic acid immobilized ferrite of Example 3, 3.6 × 10 ¹² particles (14100 ng) were mixed, and the total volume was adjusted to 100 μl with a solution of 20 mM Tris-HCl (pH 7.9), 150 mM NaCl, 5 mM EGTA, 5 mM DTT. The solvent was replaced by dialyzing this mixed solution into a solution of 150 mM NaCl, 2 mM CaCl ₂ , pH 7 for 16 hours at room temperature.

  This solution was collected, and the assembly mode of the VP1 pentamer in the substituted solvent was observed using a transmission electron microscope. The addition of dimercaptosuccinic acid-immobilized ferrite confirmed the formation of T = 1 virus-like particles that are aggregates of 12 VP1 pentameric proteins. (FIG. 4-A) Moreover, in the analysis by sucrose density gradient centrifugation, the formation efficiency was comparable to that of citrate-immobilized ferrite (FIG. 4-B).

  From the above, it can be seen that the formation of ferrite-containing virus-like particles depends on the number of ferrite particles whose surface is negatively charged.

  Furthermore, this application is based on Japanese Patent Application No. 2006-102351 filed on Apr. 3, 2006, the disclosure of which is incorporated by reference in its entirety.

Claims (25)

Viral proteins,
Fine particles containing at least one of a metal and a metal compound that are encapsulated substances;
And a virus particle-like structure that is substantially uniform in size.   The virus particle-like structure according to claim 1, wherein the fine particles have a positive or negative charge as a surface charge.   The virus particle-like structure according to claim 1 or 2, wherein at least one of the metal and the metal compound is iron, iron oxide, or ferrite.   The virus particle-like structure according to any one of claims 1 to 3, wherein the virus protein is at least one of a protein derived from a virus exhibiting an icosahedral structure and a mutant or a variant thereof.   The virus particle-like structure according to any one of claims 1 to 4, wherein the viral protein is at least one of a viral capsid protein and a mutant or a variant thereof.   The virus particle-like structure according to claim 5, wherein the capsid protein and a variant or variant thereof are at least one selected from the group consisting of VP1 capsid protein, VP2 capsid protein and variants or variants thereof. .   The virus particle-like structure according to any one of claims 1 to 6, wherein the viral protein is derived from a papovavirus genus.   The virus particle-like structure according to claim 7, wherein the viral protein is derived from a virus selected from the group consisting of SV40 virus, JCV virus and BKV virus.   The virus protein mutant or variant is a protein consisting of an amino acid sequence in which one or more amino acids are deleted, one or more amino acids are added or substituted with another amino acid in the amino acids constituting the virus protein. The virus particle-like structure according to claim 5.   The virus particle-like structure according to claim 5, wherein the virus protein variant or variant is a variant or variant produced by a gene recombination technique, a mutagenesis method, or a chemical modification method.   The virus particle-like structure according to any one of claims 1 to 10, further comprising a particle formation promoting factor that promotes self-assembly and organization of the virus protein and the microparticles. The virus according to claim 11, wherein the particle formation promoting factor is a virus particle capsid protein, DNA, RNA, or a biopolymer, an artificial polymer , a compound monomer, or a ferrite particle whose surface is coated with these. Particle-like structure.   The virus particle-like structure according to any one of claims 1 to 12, further comprising a physiologically active substance.   A preparation containing the virus particle-like structure according to any one of claims 1 to 13.   The preparation according to claim 14, wherein the preparation is an imaging agent, a diagnostic agent or a therapeutic agent for a tumor.   The preparation according to claim 15, wherein the preparation is a contrast agent for an ultrasound diagnostic imaging apparatus, a nuclear magnetic resonance imaging diagnostic apparatus or an X-ray diagnostic imaging apparatus as an imaging agent.   The preparation according to claim 14 or 15, wherein the preparation is a preparation for thermotherapy of cancer.   A composition containing the virus particle-like structure according to any one of claims 1 to 13 for introducing into the cell the fine particles contained in the virus particle-like structure. At least one of viral proteins and variants or variants thereof;
Fine particles containing at least one of a metal and a metal compound that are encapsulated substances,
A method for forming a virus particle-like structure, comprising incubating at 15 to 30 ° C at pH 5 to pH 10 in the presence of 100 mM to 500 mM monovalent cation and 2 μM to 50 mM divalent cation.   The method according to claim 19, wherein the monovalent cation is a sodium ion.   The method according to claim 19 or 20, wherein the divalent cation is a calcium ion.   The method according to any one of claims 18 to 21, wherein the concentration of the monovalent cation is 100 to 500 mM, and the concentration of the divalent cation is 1 to 5 mM. 360 parts by weight of at least one of viral proteins and variants or variants thereof, 0.01 to 100 parts by weight of fine particles having a positive or negative charge as a surface charge and containing at least one of metals and metal compounds, Mixing (I),
Dialyzing the mixture obtained in the step (I) against an aqueous solution containing a monovalent metal salt and a divalent metal salt;
A method for producing a virus particle-like structure comprising   The production method according to claim 23, wherein a weight ratio of the fine particles containing a metal or a metal compound and having a negative charge to the viral protein is 0.2 or more.   The production method according to claim 23 or 24, wherein the monovalent metal salt is a sodium salt and the divalent metal salt is a calcium salt.