JP2009249353A - Contrast agent for esr and production method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for directly observing in vivo tissue-specific deposition of DDS-related medicines or DDS carriers and presence or absence of their distribution in non-intended tissues by imaging. <P>SOLUTION: The contrast agent for ESR includes single or a plurality of objects displaying electron spin resonance (ESR) in a DDS carrier and achieves an electron spin resonance absorption line width ΔH of ≤1,000 mG in a static magnetic field of 50-500 G which is a practical magnetic field range for in vivo ESR-CT. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a contrast agent, and relates to a contrast agent for electron spin resonance (ESR) suitable for high-resolution and high-speed imaging, and a method for producing the contrast agent.

  The development costs for Kamiso have increased dramatically over the past 10 years because it is difficult to find water-soluble low molecular weight (~ 300) candidate compounds that are the mainstream of pharmaceutical candidates and the demands on the safety of pharmaceuticals have become stricter. Increasingly, reducing the cost of new drug development has become an urgent issue.

  Recently, in order to alleviate the toxicity of anti-cancer drugs, etc., and to exert an effect with an appropriate amount of drug, the upper end of a DDS (drug delivery system: drug delivery system) drug that directly reaches the cancer site to be treated In addition, several DDS anticancer drugs have reached the clinical trial phase I, II, III stage.

  When developing a new drug, it is necessary to quantitatively understand the pharmacokinetics of the drug in vivo. Conventionally, in animal experiments for drug development, each tissue distribution has been examined by analytical techniques such as high-performance liquid chromatography (HPLC), activation analysis using RI (radioisotope), and mass spectrometry. In the development of tissue-specific adherent drugs such as DDS pharmaceuticals, a diseased tissue is taken out and the distribution and accumulation of the drug in a desired diseased tissue is examined by an analytical method.

  On the other hand, there has been a remarkable progress in diagnostic imaging apparatus using contrast agents. For example, SPECT (Single Photon Emission Computed Tomography: Single Photon Emission Computed Tomography) which labels the biodistribution by labeling a radioactive element on the contrast agent. Imaging methods) and PET (positron tomography) have made it possible to obtain images of the distribution of contrast medium on the cancer site. In addition, with the development of MRI (magnetic resonance imaging) contrast agents and X-ray CT contrast agents, it has become possible to add strong contrast to morphological images. It is also possible to follow changes over time in the form of diseased tissue after drug administration by MRI or X-ray CT.

A contrast agent for ESR-CT (Electron Spin Resonance Computed Tomography) is a spin trap agent (for example, K. Takeshita et. Al., Free Radical Biology & Medicine for the purpose of imaging radical distribution in a living body). , 26 (1999) 951), the absorption line width ΔH is as wide as about 1.5 G (Gauss), and it has been difficult to image a clear contrast medium distribution. In order to overcome this drawback, a water-soluble TAM molecule (US Pat. No. 5,530,140) having an absorption line width ΔH as narrow as about 250 mG (Gauss) has been developed, and the in vivo strength distribution of the contrast agent is to some extent. It was supposed to be obtained as a clear image. In addition, a method of incorporating SPM (superparamagnetism) magnetic material into liposomes (US Pat. No. 8,905,040) is known.

In the field of ESR, as an organic crystal having a small absorption line width ΔH of about 20 mg, FA ₂ PF ₆ (difluoranthenylphosphor-hexafluoride: FA is an abbreviation of fluoranthen (fluoranthene; C ₁₆ H ₁₀ )); for example, Appl. Phys., A 30 (1983) 227) and the X phase of LiPc (lithium phthalocyanine; C ₃₂ H ₁₆ LiN ₈ ) (for example, US Pat. No. 5,112,597 and J. Phys. Chem. B104 (2000) 4047) are known. It has been. These organic crystals are produced by the electrolytic crystal growth method, and crystal growth apparatuses (called crystal growth cells) have been produced for the purpose of conducting basic research such as universities (for example, “chemistry of conductive low-dimensional materials”). ”The Chemical Society of Japan, General Chemistry No. 42, 1983, Society Publishing Center). In the case of yttrium, iron, and garnet (YIG: Y ₃ Fe ₅ O ₁₂ ) whose outer shape is close to a sphere with an outer shape of about 1 cm, the resonance frequency depends on the direction of the crystal axis. However, it is known that the absorption line width ΔH of ESR decreases to 200-500 mG if the diamagnetism of the shape effect is suppressed (for example, Uno et al., “4th Edition, Introduction to Kittel Solid Physics Volume 2” Maruzen) (1974) p. 607, FIG. 17).

US Pat. No. 5,530,140 US Pat. No. 8,905,040 US Pat. No. 5,112,597 Free Radical Biology & Medicine, 26 (1999) 951 Appl. Phys. A 30 (1983) 227 J. Phys. Chem. B104 (2000) 4047 “Chemistry of Conductive Low-Dimensional Materials”, edited by the Chemical Society of Japan, Chemistry General No. 42, 1983, Academic Publishing Center Uno et al., “4th Edition, Introduction to Kittel Solid Physics, Volume 2” Maruzen (1974), p. 607, FIG.

  In order to keep development costs low, the drug efficacy has been confirmed, but in the phase I, II, and III of the clinical trial, due to toxicity, metabolic abnormalities, excessive retention in the body, etc. On the other hand, if the drug can be packed in a DDS carrier, delivered to the tissue to be treated, and released slowly, the cost of drug development can be greatly reduced. Therefore, the DDS technology is attracting attention again. If such DDS drugs can be developed at low cost, there is a high possibility that bottlenecks in new drug development will be eliminated. In such DDS pharmaceuticals, accumulation outside the desired site is likely to cause serious problems such as toxicity and increase the risk of drug development.

  Is it possible to remove the diseased tissue and analyze the drug distribution and accumulation by using an analytical method? In order to clarify this point, it is necessary to kill animals such as mice every predetermined time after drug administration, take out each tissue, divide each tissue finely, and analyze the drug distributed in the tissue piece is there. Therefore, it is not rare that 10,000 mice are needed only by pharmacokinetic analysis of one drug candidate. (1) It has enormous labor, time and cost, and has significantly increased drug development costs. . In addition, (2) since the drug distribution of each individual to each tissue cannot be traced temporally, the drug distribution in the true sense could not be examined. Furthermore, (3) from the viewpoint of animal welfare, there were many problems in sacrificing many animals. In addition, in experiments using monkeys that have similar distribution and metabolism mechanisms to humans for the purpose of investigating the distribution and metabolism that were not problematic in mice, the distribution to each tissue can be easily examined from the viewpoint of animal protection. Since there are no, most can only measure blood concentration. In the development of tissue-specific drugs such as DDS drugs for monkeys that are often taken up as model experiments close to humans, it is necessary to kill monkeys, and the number of these must be minimized. Could not do enough.

  In the development of a tissue-specific adherent drug such as a DDS drug, the target tissue has been determined, and therefore development of an apparatus that can easily perform a screening test for the effectiveness of a DDS carrier is required. In the development of DDS drugs, the particle size of the carrier that contains the drug and the chemical structure of the carrier surface determine the tissue-specific adherence, and the carrier particle size and the chemical structure of the carrier surface are changed in various ways. The development of a method that can be used in experiments and can easily analyze tissue distribution in animals in a living state has been desired.

  On the other hand, in contrast agents for diagnostic imaging such as SPECT and PET, the signal intensity at the accumulation site and the non-accumulation site (background) is as low as about 3 times, and the misdiagnosis rate (probability of missing accumulation at a specific site) is low. It tends to be high and is difficult to use for pharmacokinetic analysis. Since the spatial resolution is poor in principle, the only way to improve the spatial resolution is to increase the radiation intensity and the accuracy of the collimator. For that purpose, it is necessary to increase the amount of radioactive elements used for the contrast agent to around the exposure limit, and even in the case of small animals, the living body will be killed. In addition, since the half-life is short, it is not easy to label DDS carriers with radioactive nuclei. Labeling itself requires an expensive synthesis facility and has the disadvantage that tissue specificity is not high because the nuclei of special molecules in the contrast agent are labeled with radioactive elements. Spatial resolution, which is the decisive factor for image quality, is about several millimeters, so that spatial resolution is poor and measurement time is long.

  In contrast, MRI contrast media and contrast media for X-ray CT do not directly image the distribution of contrast media, and tissue-specific contrast media cannot be realized. (For example, the 4th Tokyo University of Science DDS Research Center Symposium Proceedings, page 17, December 20, 2006, "Invited Lecture 3 MRI Imaging Drug Targeting" Application: Masayuki Yokoyama (Kanagawa Academy of Science and Technology).

The contrast agent for ESR-CT has a great advantage in that the distribution of the contrast agent itself can be imaged, but no tissue-specific contrast agent has been realized in the TAM molecular contrast agent. In the method of including the SPM magnetic substance in the liposome and the commercially available SPM contrast agent, tissue-specific adherence cannot be realized as the MRI contrast agent, and the absorption line width for the contrast agent for ESR-CT is 60- Since it is an order of magnitude as large as about 1240G (Fe ₃ O ₄ etc.), a clear distribution image could not be obtained, and it was completely unsuitable as an ESR contrast agent.

Conventional crystal growth cells for producing organic crystals are crystal growth cells whose main purpose is to search for new substances and to supply crystals to physical experiments. Therefore, in order to produce nanoprobe agents industrially, a large amount of FA ₂ PF ₆ crystal and LiPc crystal (X phase) are required, and when creating with a conventional crystal growth apparatus, there is no choice but to arrange a large number of crystal growth cells in parallel, and workability (ease of work and work time) ), A large cell installation area and raw material utilization efficiency were totally unsuitable.

Conventionally, a ferromagnetic material such as ferrite (Fe ₃ O ₄ or the like) has to have a size of several nanometers to make it an SPM magnetic material, and it is difficult to pulverize the bulk size to make it small, and it is exclusively chemical. It has been formed by methods (coprecipitation method, etc.). However, a magnetic material formed by a chemical method has poor crystallinity, an absorption line width is widened, and it is difficult to obtain a large magnetization.

  In the development of tissue-specific adherent drugs such as DDS pharmaceuticals, have they been (1) delivered to the desired diseased tissue from the stage of animal experiments? (2) Is there any accumulation outside the desired organization? Clarifying this point is the key to the practical application of pharmaceuticals. At the stage of animal experiments, it is not a method of killing animals and analyzing their distribution to tissues, but tissue-specific adherence in a living state The realization of a method for analyzing the drug is a major issue for efficiently developing DDS pharmaceuticals.

  The main object of the present invention is to provide an image imaging method for in vivo tissue-specific adherence (distribution) of DDS pharmaceuticals and DDS carriers and the presence or absence of distribution to an unexpected tissue without killing animals in the course of drug development. To provide a means of direct observation.

  The contrast agent of the present invention includes an ESR signal source (hereinafter referred to as an ESR object) in a structure (hereinafter referred to as a DDS carrier) whose outermost surface is composed of a hydrophilic group, and a biocompatible aqueous solution, for example, It is a contrast medium dispersed in physiological saline, and the above object can be achieved by administering this contrast medium to a living body and taking an ESR-CT image.

  When a DDS drug is prepared by encapsulating a drug in a DDS carrier having the same particle size and chemical structure of the outermost surface of the DDS carrier used in this contrast agent, if the distribution of the contrast agent is known, Is the distribution of the DDS carrier that has been incorporated, and (1) was it delivered to the desired lesion tissue? (2) Is there any accumulation outside the desired organization? This can be discriminated visually.

  When the contrast agent of the present invention is used, in drug development, the particle size and surface structure of DDS can be changed variously to easily image the change in the site of attachment in the living body. The important point here is that the DDS carrier that confines the drug and the DDS carrier that confines the ESR object have the same particle size and chemical structure of the outermost surface of the DDS carrier. It does not have to be the same.

  When a living body such as a small animal or a human is targeted, dielectric loss becomes a problem when the frequency increases during microwave irradiation, and the microwave cannot enter the center from the living body surface. In the case of humans, it is known that microwaves enter the whole body up to 200 MHz, but the physical mechanism is not understood in detail. The upper limit of the resonance magnetic field of ESR-CT is considered to be about 500 G (corresponding to a frequency of 1.4 GHz). On the other hand, if the resonant magnetic field is lowered, it easily enters the living body and the problem of dielectric loss is eliminated. However, since the sensitivity is deteriorated, the lower limit of the resonant magnetic field is about 50 G (corresponding to a frequency of 140 MHz). Eventually, in biological applications such as small animals and humans, the resonance magnetic field is used in the range of 50G to 500G, and it is important that clear ESR-CT images that can discriminate tissue specificity within this magnetic field range can be taken. It is. For this purpose, it is desirable that the spatial resolution of the distribution is at least 1 mm or less.

  Means for realizing the present invention will be described in more detail. Single or multiple objects (ESR objects) exhibiting electron spin resonance (ESR) are included in the DDS carrier, and a practical magnetic field range of 50 G (Gauss) to 500 G (Gauss) in a biological ESR-CT The object of the present invention can be achieved by realizing a contrast agent for ESR capable of reducing the electron spin resonance absorption line width ΔH to 1000 mG or less under a static magnetic field. Since the particle size of the DDS carrier and the chemical structure of the outermost surface determine the presence or absence of tissue specificity, when developing a DDS pharmaceutical, the particle size of the DDS carrier and the chemical structure of the outermost surface are used as parameters. The object of the invention is achieved by including an object exhibiting an ESR signal having the above properties in the DDS carrier. As for the particle size of the DDS carrier, when considering biological application, when administering directly to the vascular system, or when administering by injecting into tissue, when trying to circulate the vascular system through the capillaries, It is often used at about 1 μm or less. It is known that a DDS carrier having a particle size of approximately 400 nm or less is effective when a drug is to be carried to a cancer tissue through a pore of about 1 μm generated in the tube wall of a new blood vessel such as a solid cancer. On the other hand, in the case of a DDS carrier used as an oral drug, the particle size restriction is greatly relaxed. For example, when there is an abnormality in a specific intestinal wall and it is applied to the wall, the particle size may be about 100 to 1000 μm.

In the case of ESR-CT or MRI, when the spatial resolution a is evaluated, the upper limit is expressed by the ratio between the absorption line width ΔH and the gradient magnetic field strength G.
a = ΔH / G (1)

  If the gradient magnetic field strength G is increased, the spatial resolution can be improved in principle if the radical has a narrow absorption line width ΔH, but there are some limitations in reality. One of the major problems is that when the gradient magnetic field strength G is increased, the necessary gradient magnetic field forming power becomes extremely large.

  As disclosed in the ESR-CT apparatus disclosed in Japanese Patent Application Laid-Open No. 2006-245597, the gradient magnetic field strength G has a practical limit of about 100 G / cm even when a zoom-up function is provided and only a local region is enlarged and imaged. In order to guarantee a spatial resolution of 1 mm or less, the absorption line width ΔH needs to be about 1000 mG or less from the equation (1). Desirably, if it is 300 mG or less, the gradient magnetic field strength G becomes about 30 G / cm, and a clear image can be obtained without imposing a large burden on the ESR-CT apparatus.

As a specific configuration of such an ESR contrast agent, two basic structures are shown below.
The first is that the electron spin resonance absorption line width ΔH of the ESR object itself is 1000 mG or less and the resonance magnetic field of the ESR object changes depending on the angle between the ESR object and the static magnetic field (crystal axis anisotropic If the average resonance magnetic field is defined as 1/2 of the sum of the maximum resonance magnetic field and the minimum resonance magnetic field, (maximum resonance magnetic field−minimum resonance magnetic field) is 1/500 or less of the average resonance magnetic field. The ESR object having a very small resonance magnetic field anisotropy is employed.
(Maximum resonance magnetic field−minimum resonance magnetic field) ≦ average resonance magnetic field / 500
= (Maximum resonance magnetic field + minimum resonance magnetic field) / 1000 (2)

  When a group of ESR objects that cause magnetic resonance can be regarded as a group in which individual ESR objects are randomly arranged with respect to an applied static magnetic field (the contrast agent for ESR of the present invention is also a group of such ESR objects). In the assembly of ESR objects, the crystal axes are expected to be randomly distributed with respect to the magnetic field, so that the resonance absorption line width ΔH is the maximum resonance magnetic field and the minimum resonance magnetic field as a whole. Is observed as a peak having a single absorption line width ΔH. In this case, since the resonance absorption line width ΔH (difference between the maximum resonance magnetic field and the minimum resonance magnetic field) is proportional to the applied magnetic field, it can be called the absorption line width due to the applied magnetic field effect.

In general, the following relationship exists between the resonance frequency ν of electron spin resonance and the applied static magnetic field H.
hν = g · e / (2Mc) · h / 2π · H (3)

  Here, M is the mass of the target electron, e is the unit charge, c is the speed of light, h is the Planck constant, and the sign (±) of the charge is not particularly considered because it does not matter within the scope of the present invention. g is a quantity inherent to an electron called a g value, and in a free electron, g = 2.0023. It is known that the g value generally shifts from the value of free electrons for electrons in crystals and electrons in molecules.

The resonance magnetic field anisotropy described above can be rephrased as the g value anisotropy Δg / g being 1/500 or less.
Δg / g ≦ 1/500 (4)

  In the presence of g-value anisotropy, the resonance frequency distribution due to the crystal orientation expands in proportion to the strength of the applied magnetic field, and in the aggregate of ESR objects in which the crystal orientation is distributed in a random direction with respect to the applied magnetic field. Behaves as a substance having a resonance absorption line width ΔH according to the g value anisotropy Δg / g (contrast agent for ESR of the present invention). The contrast agent for ESR of the present invention has an electron spin resonance absorption line width ΔH of itself as an ESR object of 1000 mG or less if the product of the applied static magnetic field H and the g value anisotropy Δg / g is 1000 mG or less. It is. In this contrast agent, the absorption line width is proportional to the applied magnetic field at a high magnetic field, and at a low magnetic field such as 50-500G, the contribution of the magnetic field proportional portion to ΔH is reduced and the absorption line width is narrowed. In the limit of the low magnetic field, the absorption line width inherent to the ESR object constituting the contrast agent for ESR is narrowed.

In the field of ESR as a material having such properties, in the case of FA ₂ PF ₆ (difluoranthenyl phosphorhexafluoride) crystal or X-phase Li—Pc (lithium phthalocyanine) crystal, the absorption line width ΔH is large in bulk crystal (approximately 1 mm or more in particle size). It was known that there was a very narrow and slightly g-valued crystal anisotropy Δg (Δg / g≈1.8 × 10 ⁻⁴ ). However, in such an organic crystal, no method has been found for forming fine particles having a particle diameter of 1 μm or less, and it has not been proved whether or not this bulk property is maintained when the particle diameter is 1 μm or less. It was. Further, these crystals are hydrophobic, and no method for dispersing them in an aqueous solution has been found. In producing the contrast agent, a large amount of FA ₂ PF ₆ crystals and X-phase Li—Pc crystals are required, but a method for mass production of these crystals has not been known. These will be described in detail in specific embodiments of the present invention.

The surface of the FA ₂ PF ₆ crystal or the Li—Pc crystal is hydrophobic, but the contrast agent of the present invention can be realized using a hydrophilic ESR object. Li-TCNQ (7,7,8,8-tetracyanoquinonedimethane), K-TCNQ, and Na-TCNQ crystals are known as hydrophilic ESR objects having an absorption line width as narrow as 300 to 1000 mG. These powder crystals are finely divided and dispersed in an aqueous solution, and the contrast agent for ESR of the present invention can be realized from this aqueous solution. This method will be described in detail in the embodiments.

The second method for realizing the contrast agent for ESR of the present invention is a method using a special case shown below in the superparamagnetic state (SPM). A magnetic moment M forming a single magnetic domain of a ferromagnetic material or a ferrimagnetic material is in a state of moving at a certain time scale τN from the direction of the easy axis of magnetization determined by the crystal structure that has been fixed by thermal energy. If we define the isotropic energy as K ₁ , the volume of the magnetic material as V, the absolute temperature as T, and the Boltzmann constant as k _B ,
K ₁ V ≦ 25k _B T (5)
The time when the condition is satisfied is generally called SPM. The time scale τN is a time shorter than about several tens of seconds and is called a nail time.
τN to exp (KV / k _B T) / f ₀ (6)
F ₀ is a frequency of about 10 ⁹ to 10 ¹⁵ (1 / s). In other words, when KV = 25 k _B T in Equation (5), the magnetization is reversed in a time on the order of τN to 72 s-72 μs.

The approximate value of the maximum diameter (nm) that realizes SPM (superparamagnetism) in four typical ferromagnets was obtained by spherical approximation 2 · (3V / 4π) ^1/3 at room temperature (T = 300K). Table 1 shows the results. YIG (yttrium, iron, garnet; Y ₃ Fe ₅ O ₁₂ ) crystals have a small magnetic anisotropy among ferromagnetic materials, and therefore have a grain size that realizes SPM compared to ferrite (Fe ₃ O ₄ ). The feature is as large as 68 nm. The value of the particle size that realizes SPM is calculated from the anisotropic energy K ₁ and the volume V of the magnetic material using the formula (5) (however, the temperature is room temperature).

  When this diameter is called the SPM critical diameter, the critical diameter increases as the magnetic anisotropy decreases. When administered to a living body, the temperature is 37 ° C. (310 K in absolute temperature), but only changes about 1% from the estimated value at room temperature (T = 300 K). For reference.

In SPM, if it becomes smaller than the maximum particle size shown in Table 1, according to formulas (5) and (6), a huge magnetic moment can move around at high speed due to the surrounding thermal energy. It has been applied to contrast agents. However, conventional magnetite (Fe ₃ O ₄ ) and hematite (γFe ₂ O ₃ ) have been used as contrast agents for MRI having a particle diameter of 1-2 nm, but the ESR absorption line width of SPM fine particles dispersed in an aqueous solution. When ΔH was measured, even a narrow one was 60G (Gauss) and it was overwhelmingly too wide to be used for the contrast agent for ESR, and it was not practical as a contrast agent for ESR-CT.

  On the other hand, it was technically difficult to bring the shape of oxide crystals such as bulk YIG closer to a sphere so as not to cause a shape effect (diamagnetic effect). However, the rapid progress of wet milling methods has made it possible to uniformly form spherical beads with a small particle size, and nano-particles with a size of several nanometers can be produced even by the milling method. .

YIG crystals known to be cubic and have a small anisotropic energy K ₁ exhibit superparamagnetism (SPM) with a relatively large particle size of 68 nm or less compared to other ferromagnetic materials. While examining the ESR absorption line width ΔH when the YIG particle size is further reduced, when the median particle size reaches 4 nm, the YIG nanoparticles are dispersed in an aqueous solution of 2 GHz. The following absorption line width was found to be 1000 mG or less.

This phenomenon was found to be a physical phenomenon similar to motional narrowing, in which the proton nuclear magnetic resonance (NMR) absorption line width dramatically decreases when ice is dissolved in water and then changed from solid to liquid. This phenomenon should be called “rotational narrowing”, and minute YIG nanoparticles collide with molecules in an aqueous solution and randomly rotate. Then, the magnetic anisotropy field Ha in the YIG crystal rotates at random, and the local magnetic field fluctuates rapidly with respect to the magnetic moment of the YIG nanoparticle, so that it behaves as if there is no absorption line width. It was found that ΔH is a very small phenomenon. In the case of a cubic system such as a YIG crystal, the magnetic anisotropy field Ha is
_{Ha = K 1/3 ... (} 7)
The relationship represented by is known. If the rotational magnetic ratio of the electrons constituting the SPM is γe (absolute value is described below), the magnetic anisotropic magnetic field Ha, and the rotational relaxation time of the YIG nanoparticle are τB, the condition for causing rotational narrowing is
γe · Ha · τB <1 (8)
It was found that This is a condition that the rotation angle of the spin by the magnetic anisotropy magnetic field Ha is 1 radian or less during the correlation time τB in which the angle of the particle changes.
The condition for obtaining a clear image which is the object of the present invention is that ΔH ≦ 1000 mG with respect to the absorption line width ΔH, and the absorption line width ΔH is when rotational narrowing occurs.
ΔH = (γe · Ha · τB) · Ha (9)
It was found that it is expressed. Therefore, the contrast agent
(Γe · Ha · τB) · Ha ≦ 1000 mG (10)
When the above condition is satisfied, the contrast agent of the present invention is obtained.

Here, the rotational relaxation time τB is obtained by using the volume of the YIG nanoparticle as V, the viscosity coefficient η of the solution in contact with the YIG nanoparticle, the absolute temperature T, and the Boltzmann constant k _B.
τB = 3Vη / k _B T (11)
It is a time constant represented by It was found that the volume V of the equation (11) can be approximated by the following equation, where d is the median particle size of the YIG nanoparticles dispersed in the solution.
V = 4π (d / 2) 3/3 ... (12)

  It has been found that the absorption line width is 1000 mG or less when dispersed in an aqueous solution by using a YIG crystal as such a superparamagnetic material and a crystal median particle size d of 5.5 nm or less. . The reason why such a narrow absorption line width ΔH is obtained with the YIG dispersion is that, in a ferromagnetic material or a ferrimagnetic material, the magnetic anisotropy field Ha of YIG is extremely small, and thus a relatively large crystal median. This is because ΔH was as small as 1000 mG at a particle size of 5.5 nm.

In fact, in a ferromagnetic material or a ferrimagnetic material having a magnetic anisotropy field Ha that is one digit or more larger than that of ferrite (Fe ₃ O ₄ ), ΔH is as large as 50 G even if the crystal median particle size is 2 nm.

  Superparamagnetic YIG crystals tend to have narrower absorption line widths in near-spherical nanoparticles whose ratio of median minimum length to maximum length (maximum length / minimum length) is 1.5 or less.

  In the case of a TAM molecule, the absorption line width ΔH is as large as about 4.2 G when solid. However, in the case of a TAM molecule in an aqueous solution, since it moves randomly due to collision from surrounding molecules, ΔH is 300 mV as a result. Narrow to the extent. Therefore, the present invention can be constructed by a method of confining the aqueous TAM molecule solution in a micelle with a high concentration. The invention for TAM molecules is described in detail in the examples.

The contrast medium configured as described above has a single peak from the standpoint of ESR absorption lines, and is optimal as a contrast medium for ESR-CT. This is because when the contrast agent has a plurality of peaks, a specific peak is monitored and imaged, so that there is a drawback that the image construction method simply cannot be performed. The minimum requirement for an ESR object is that it can exist stably in the living body for at least 1 hour, and the practical means that can be avoided even if the toxicity is negligible or can be easily found.
Since the ESR object described above has this property, the object of the present invention can be achieved.

  When the contrast agent of the present invention is used, in drug development, the particle size and surface structure of DDS can be changed variously to easily image changes in the adherent site in the living body. Become a support tool for designing.

  According to the present invention, it is possible to image the biodistribution of a DDS carrier in a living state without killing a small animal. It is also useful as a tool for examining changes. (1) Was the drug developer designed to the desired DDS carrier surface structure and particle size and delivered from the animal experiment stage to the desired diseased tissue (or target site)? Will it accumulate outside the desired organization? This point can be clarified by image imaging. (2) By selecting a substance having an absorption line width of 300 mG or less, the distribution from the whole living body can be rapidly imaged within 15 minutes with a spatial resolution of 1 mm or less. (3) Was the nanoprobe and the DDS drug administered simultaneously using the substance, and was it delivered to the desired diseased tissue (or target site) of the DDS drug? Will it accumulate outside the desired organization? This point can be clarified by image imaging.

  In the development of DDS pharmaceuticals, the target organization has been determined, so it is extremely effective for screening tests for the effectiveness of DDS carriers. It is particularly effective to determine whether or not to adhere to an unexpected part by an image. In addition, the ESR-CT apparatus (Japanese Patent Laid-Open No. 2006-245597) using a wide gap permanent magnet is very effective because it is a compact size and cheaper than PET and MRI.

  In the case of humans who can only observe blood dynamics, it is virtually impossible to examine the distribution of drugs accumulated in each tissue, especially in the case of drugs of DDS type, their specific adherence and unexpected adherence. The present invention which can be analyzed by image diagnosis is highly useful.

  In the case of a contrast agent using YIG, the absorption line width changes when the environment surrounding YIG changes. By analyzing this change, the interaction between the contrast agent deposition site and biological tissue and the DDS carrier is analyzed. Now we can provide a means to do it.

  Although not the direct object of the present invention, the amount of contrast medium can also be evaluated by taking out a living tissue administered with the contrast medium and performing ESR evaluation.

Embodiments of the present invention will be described below with reference to the drawings.
First, the basic configuration of the present invention will be described. 1A to 1C are schematic views showing the basic structure of a contrast agent for ESR according to the present invention.

FIG. 1A shows a lipophilic group 3 of a surfactant such as phospholipid on a single ESR object 1 (in this example, FA ₂ PF ₆ microcrystal, LiPc (X phase) microcrystal, superparamagnetic YIG nanoparticle, etc.). Is adsorbed on the ESR object 1, and the hydrophilic group 2 of the phospholipid surfactant is arranged on the surface side of the DDS carrier. In this case, the surface of FA ₂ PF ₆ crystal and LiPc (X phase) microcrystal is not problematic because it is strong in hydrophobicity (the same as lipophilicity), but in the case of superparamagnetic YIG nanoparticles, the hydrophobicity is increased. As shown in Examples, surface treatment is performed. This structure is effective when the surface of the ESR object 1 is inherently hydrophobic or can be modified to be hydrophobic.

  FIG. 1B shows an ESR object 8 (in this example, superparamagnetic YIG nanoparticle, Li-TCNQ microcrystal, K-TCNQ microcrystal, Na-TCNQ microcrystal, TAM molecule, etc. whose surface is modified to be hydrophilic). , Dispersed in water-soluble solution 7 and made into liposome (consisting of a double membrane of outer phospholipid hydrophilic group 21, lipophilic group 31 and inner phospholipid hydrophilic group 21 ', lipophilic group 31') It is a structure included in In this case, the superparamagnetic YIG nanoparticle whose surface is modified to be hydrophilic can have the structure shown in FIG. 1A.

  In this structure, Li-TCNQ, K-TCNQ, and Na-TCNQ crystals are pulverized in physiological saline and confined in liposomes in a fine particle state. The structure shown in FIG. 1B can also be realized by confining a high-concentration TAM molecule aqueous solution in the liposome.

FIG. 1C shows an ESR object 10 (in this case, superparamagnetic YIG nanoparticles, FA ₂ PF ₆ microcrystals, LiPc (X phase) microcrystals, etc.) dispersed in an oily solution 9 such as soybean oil, and phospholipids such as lecithin. It is the structure which was included with surfactant 11 such as.

FA ₂ PF ₆ crystal and LiPc crystal (X phase) are paramagnetic from the standpoint of ESR and have a slight g value of crystal anisotropy Δg (Δg / g≈1.8 × 10 ⁻⁴ ). To do. However, in the ESR-CT for living bodies, since the resonance magnetic field is as low as 50 to 500 G, Δg hardly affects the ESR image. Therefore, the absorption line appears as a single peak, and in the process of forming nano-particles, it suppresses deterioration near the surface of the fine particles and maintains the properties of the bulk crystal state (g value and absorption line width ΔH) for ESR-CT. Applicable to contrast media.

  On the other hand, the bulk crystal of YIG crystal is a ferromagnetic material, and from the standpoint of ESR, has a strong anisotropy Δg, and when a static magnetic field is applied to a fixed single crystal (single magnetic domain), Depending on the relative orientation of the azimuth and the static magnetic field, the absorption frequency line width ΔH spreads to 270 G (X band) in a fine powder sample having a particle size of several microns with variously different crystal orientations and different resonance frequencies. Even in the case of the low resonance magnetic field 70G used in ESR-CT for living bodies, the absorption line width ΔH is about 6G. However, as described above, the absorption line width can be remarkably reduced by the “rotational narrowing” effect in the state of being dispersed in the solution by using SPM and further reducing the particle size. Will be described in more detail.

FIG. 1D is an explanatory diagram for explaining the second embodiment of the present invention in detail. The median particle diameter d and the low magnetic field (50G-500G) when superparamagnetic YIG nanoparticles are dispersed in a solution having a viscosity coefficient η. The relationship of ESR absorption line width ΔH in FIG. η ₀ is a viscosity coefficient of pure water at 37 ° C. of 7.0 × 10 ⁻³ Pa · s, and 37 ° C. shows a typical value of the body temperature of mammals such as mice. The viscosity coefficient η of living animal tissue, body fluid, and blood is distributed between η ₀ and 10η ₀ , and when a contrast agent is administered, the viscosity during this time becomes a problem. However, if the particle size starts to become smaller than about 0.8-1.5 nm, it will start to become several times the crystal unit volume of YIG, start to be strongly influenced by the surface, and the influence of the production method will become significant and small. On the other hand, since the absorption line width starts to increase rapidly, a reproducible manufacturing method becomes difficult. In the case of YIG nanoparticles, the hatched area 50 in FIG. 1D is the most suitable area as a contrast agent.

  However, this region 50 is a case where a living organism whose biological solution temperature is 37 ° C. is considered, and the contrast agent of the present invention is also effective when extracting the tissue and examining the content by ESR. The particle size of YIG nanoparticles can be used up to about 10 nm.

  One of the features of YIG nanoparticles is that the absorption line width becomes very wide when the YIG nanoparticles are adsorbed on a living tissue and cannot move thermally, and the time change of the ESR-CT image is followed. Can capture the change.

Next, the basic form of the contrast agent production method of the present invention will be described. The first is the use of an electrolytic crystal growth apparatus that greatly improves the yield (crystal amount / crystal raw material) of the FA ₂ PF ₆ crystal and the LiPc crystal (X phase), which are electrolytic transfer organic crystals. The electrolytic crystal growth method is a method in which a current is passed through a solution in which a crystal raw material is dissolved to grow a crystal on an anode. As shown in FIG. 2A, the present inventor uses a vertical crystal growth apparatus (mass production type crystal growth cell) in which a plurality of anodes 65 are arranged on the circumference and a common cathode 62 is arranged at the center of the circle. By devising, the three points of workability (ease of work and work time), large cell installation area, and raw material utilization efficiency were remarkably improved. As shown in FIG. 2B, in the conventional crystal cell, an anode 72 is arranged in a glass triangular flask 71, a cathode 74 is arranged in a cylindrical test tube 73, both are connected by an electrolytic solution, and a cathode generated by an electrolytic reaction. A fill 75 is formed so that foreign matter from the anode does not diffuse to the anode side, and a vacuum cock 76 is formed so that the crystal cell can be evacuated and replaced with nitrogen (see Non-Patent Document 4).

  As shown in FIG. 2A, the newly developed crystal cell has a cylindrical filter-equipped cathode container 61 concentrically arranged in a cylindrical glass container 60 and a common cathode 62 at the center of the circle. A lid of a circular Teflon 63 is bonded to the upper surface of the cylindrical glass container 60 with silicon grease or the like, and is pressed by a pressure jig (not shown). A vacuum pulling window 64 is attached to the Teflon lid 63 so that the inside of the container can be replaced with nitrogen. A plurality of anodes 65 are arranged concentrically outside the cathode container 61 with a columnar filter. In the example of FIG. 2A, 12 anodes are arranged at equal intervals. The reason is that a high crystal yield and a high-quality crystal can be obtained by arranging the anode 65 and the cathode 62 to have the same electric resistance. In the figure, the dotted line portion shown between the cylindrical glass container 60 and the filtered cathode container 61 represents an anode region 67 per anode, and physically separates the electrolytic solution using a partition plate. You can also. As the filter 66, P16 having a pore size of 10-16 μm is typically used.

The cathode container 61 with a cylindrical filter can be roughly classified into a horizontal type (FIG. 2C) where the filter 66 is attached to the side and anode positions of the cathode container, and a bottom type (FIG. 2D) attached to the bottom of the container. The horizontal type is excellent for suppressing the diffusion of foreign substances that can be formed on the cathode, but the bottom type is excellent for ease of manufacturing the device. FIG. 2A shows an example in which a horizontal die attached to the anode position is installed.
Table 2 shows the difference between the mass-produced crystal cell shown in FIG. 2A and the conventional cell shown in FIG. 2B.

As can be seen from Table 2, the crystal cell of the present invention has a crystal yield (FA ₂ PF ₆ crystal amount obtained with the same FA amount) of about 2.1-2.9 compared to the conventional cell shown in FIG. 2B. Double the amount of crystal of FA ₂ PF ₆ can be obtained. Actual crystal growth will be described in detail in Example 1.

Another basic form of the present invention relating to the manufacturing method relates to a method for producing a DDS carrier structure constituting the contrast agent schematically shown in FIGS. 1A-1C. In the following, the FA ₂ PF ₆ crystal will be described as an example, but this method can be applied to other ESR objects used in the present invention.

  FIG. 3A is a schematic diagram of the apparatus, and FIG. 3B is a schematic diagram of the process. As shown in FIG. 3A, a surfactant such as phospholipid is dissolved in an insulator flask or the like using an organic solvent such as chloroform or cyclohexane, and the solvent is evaporated while rotating while applying nitrogen gas. A thin film of a surfactant such as lipid is formed and dried. Next, the solution containing the ESR object is added, and the solvent is evaporated while rotating while applying nitrogen gas, and a thin film containing the ESR object is formed on the thin film of a surfactant such as phospholipid. The process of forming a thin film of a surfactant such as phospholipid is repeated to form a thin film of a surfactant such as phospholipid on the thin film containing the ESR object. Thereafter, physiological saline or the like is added to a container such as this insulator-type flask, the three-layer film on the flask wall is peeled off, and a micelle-type contrast agent raw material is produced with an ultrahigh-speed emulsifier. Thereafter, in order to reduce the particle size, a step of dispersing in the nano-particles by a wet mill method is performed, and to increase the concentration, centrifugation is performed, and an ESR contrast agent is prepared through a filter filtration process for uniforming the particle size. .

Hereinafter, the present invention will be described in more detail through examples.
[Example 1]
First, an example in which FA ₂ PF ₆ single crystal is used will be described. Table 3 shows three specific dimensions for the mass-produced crystal growth cell (FIG. 2A) produced this time. The design parameters in Table 3 correspond to FIG. 2A.

Using this crystal growth apparatus, crystals were produced under the crystal growth conditions shown in Table 4. FA (fluoranthene) as a crystal raw material was recrystallized three times using ethanol to remove impurities. Since TBA-PF ₆ has a relatively high purity, a single recrystallization with ethanol was sufficient. About 0.4 to 1.5 g of FA ₂ PF ₆ single crystal could be secured in one week per crystal growth cell.

Actual yield data is shown in FIG. The horizontal axis of the figure represents the electric resistance at room temperature between the cathode 62 and one anode 65 in the crystal cell of FIG. 2A, and chloroform was used as the solvent. In the figure, ○ indicates mass production cell No. 3 (filter pore size 160 μm), Δ indicates mass production cell No. 3 (filter pore size 16 μm), ■ indicates mass production cell No. 2 (filter pore size 160 μm), and ◆ indicates mass production cell No. 2 This is a case where (filter pore diameter of 16 μm) is used. Actually, in order to further increase the yield, about 10 of the present crystal growth cells were arranged in parallel to mass-produce FA ₂ PF ₆ crystals. In actual crystal growth, Unit 2 with a high crystal yield was used. There was no significant difference in yield between the horizontal filter (FIG. 2C) and the bottom filter (FIG. 2D), so the bottom filter (pore diameter 16 μm) was used in the following examples.

The FA ₂ PF ₆ crystal produced in this way is a needle-like crystal having a length of several mm and a width of about 0.5 mm, or a plate-like crystal having a width of about 1.5 mm. The absorption line width at 200 MHz was about 20 mG.

Next, a procedure for manufacturing a micelle structure will be disclosed. Table 5 shows typical examples of conditions for preparing the FA ₂ PF ₆ crystal micelle nanoprobe prepared this time.

First, the basic structure shown in FIG. 1A was prepared this time, in which a phospholipid was formed on the surface of the FA ₂ PF ₆ crystal and dispersed in physiological saline (DPPC micelle). In the case of a DPPC micelle, it is prepared under the condition 0 of Table 5, and L-α dipalmitoylphosphatidylcholine (L-αdipalmitoylphosphatidylcholine: DPPC molecular weight 734.0) is used as a phospholipid. The specific production procedure will be described below. The measurement result of the particle size distribution in a state dispersed in physiological saline was as shown in FIG. 5, and the median particle size was 0.85 μm.

  Furthermore, as shown in FIG. 6A, a structure (mouse with PEG) in which PEG (polyethylene glycol) 6 is arranged on the surface was examined. The micelle structure that modifies the surface of the PEG chain aims to avoid predation from Kupffer cells present in the liver and spleen, increase the retention in blood, and increase the amount accumulated in cancer tissue. In the case of micelles with PEG, the micelles were prepared under several conditions (conditions 1-4 in Table 5) with different mixing ratios.

Phospholipids based on L-α distearoylphosphatidylethanolamine having two alkyl groups C ₁₇ H ₃₅ constituting stearic acid (L-α distearoylphosphatidylethanolamine, DSPE: C ₄₁ H ₈₂ NO ₈ P, molecular weight 748.1) PEG-DSPE with PEG (SUNBLIGHT DSPE-20CN: Nippon Oil & Fats) was selected. Cholesterol (C ₂₇ H ₄₅ OH, molecular weight 386.66: abbreviated as CS) is used as a surfactant L-α dipalmitoyl phosphatidylcholine (for the purpose of improving the adsorptivity of PEG-attached phospholipids to the surface of FA ₂ PF ₆ single crystals. L-αdipalmitoylphosphatidylcholine: DPPC, molecular weight 734.0). In this example, it is called phospholipid with PEG, and is distinguished from phospholipid of DPPC alone.

Next, using the low-temperature drying process described with reference to FIGS. 3A and 3B, a three-layer structure in which FA ₂ PF ₆ crystals are wrapped with a phospholipid thin film on the flask wall surface is prepared. Specifically, (1) phospholipids only on the wall surface of the flask, (2) phospholipids and FA ₂ PF ₆ single crystal powder are dried together, (3) phospholipids only on the wall surface. Step on. In this case, the FA ₂ PF ₆ single crystal can realize the following process even if a few millimeters of crystal obtained by the electrolytic crystal growth method is used as it is. This is because the FA ₂ PF ₆ single crystal is soft and is therefore torn into fine particles by an ultra-high speed emulsification process. In this process, an evaporator (manufactured by Tokyo Rika Kikai Co., Ltd .: model N-N1-R, 1000 mL) is installed in a program thermostat (manufactured by Yamato Kagaku: model IN602W), and the thermostat is kept at 10 ° C. and dried at low temperature. Went. The accuracy of the set temperature and the temperature distribution in the thermostat are ± 0.3 ° C. and ± 1 ° C., respectively. The FA ₂ PF ₆ single crystal was produced by a mass production crystal growth cell (FIG. 2A) developed for studying the production process of nanoparticles.

As a solvent used for low-temperature drying, low-temperature (10 ° C.) cyclohexane that does not deteriorate the FA ₂ PF ₆ single crystal was examined. The phospholipid powder did not dissolve in cyclohexane, but dissolved when a solution dissolved in a small amount of chloroform was added to cyclohexane. Since the boiling point of chloroform at 61 ° C. (1 atm) is lower than the boiling point of cyclohexane at 81 ° C. (1 atm), the evaporator evaporates faster than cyclohexane, so that chloroform does not remain. Chloroform deteriorates the FA ₂ PF ₆ single crystal, so it was set to 5% which is negligible compared to cyclohexane. After completion of the low temperature drying process, the flask was sealed and stored in a -20 ° C freezer. The low-temperature dry flask is swelled with 30 mL of physiological saline, and the flask is vortexed for 10 minutes and stirred well. The resulting solution is transferred to a container (50 mL) and rotated at 20000 rpm using an ultra-high speed emulsifier. A structure in which phospholipid was covered with FA ₂ PF ₆ crystal fine particles was realized. Phospholipids that have failed to incorporate FA ₂ PF ₆ crystal fine particles form empty micelles.

The sample prepared in this way has a FA ₂ PF ₆ crystal grain size of about 1 μm, which is large as a contrast agent. As already described, the particle size distribution at this stage is good and has a sub-micron particle size as shown in FIG.

In the case of a contrast agent for solid cancer, it is known that only fine particles having a particle size of 0.4 μm or less accumulate through pores of new blood vessels that can form solid cancer. Many anticancer drugs aim at drugs that can selectively attack cancer sites using this property. Therefore, the ESR contrast agent of the present invention needs to be further finely divided. Therefore, by a bead mill using FA ₂ PF ₆ crystal fine particle solution and ZrO ₂ having a particle diameter of 0.1 mm as beads by a wet bead mill method, fine particles are formed for 60 minutes at a rotation speed of 2300 rpm, and FA ₂ PF ₆ having a median diameter of 300 nm. A stock solution of crystalline micelle contrast agent was prepared. However, the FA ₂ PF ₆ crystal concentration contained in this solution did not reach the target concentration of 3.8 mg / mL or higher. Assuming that the target 3.8 mg / mL is about 0.1-1% of the dose accumulated in the cancer site using an ESR-CT device (Japanese Patent Laid-Open No. 2006-245597) using a wide gap permanent magnet. Thus, it is determined from the sensitivity of the device.

Therefore, centrifugal concentration was performed at 14300 rpm for 5 minutes, the supernatant was removed, and a 5.3 mg / mL FA ₂ PF ₆ crystal micelle contrast agent could be prepared. An example of the particle size distribution at that time is shown in FIG. 6B. FIG. 6C shows measured values of FA ₂ PF ₆ crystal concentration measured by 200 MHz ESR. In the figure, ♦ indicates the measurement result before centrifugal concentration, and ◯ in the figure indicates the measurement result in the concentrated state after centrifugal concentration. The horizontal axis in the figure corresponds to the lot numbers produced under the production conditions of 5CLM-1, 5CLM-1 / 2 and 5CLM-1 / 4 in Table 5. Incidentally, the FA ₂ PF ₆ crystal concentration (nominal value) under the micelle preparation conditions in Table 5 is the amount of FA ₂ PF ₆ crystal administered in the first solution, which is considerably different from the actually measured concentration. The reason is that part of the FA ₂ PF ₆ crystal is decomposed during the preparation of the contrast agent and an ESR signal cannot be output.

The prepared FA ₂ PF ₆ single crystal micelle contrast agent (referred to as micelle with PEG), DDPC micelle having a median particle size of 0.85 μm shown in FIG. 5 and two types of contrast agents are prepared. 3) Intravenous injection of 0.2 mL into a cancer-bearing mouse (cancer type is colon 26), and accumulation of FA ₂ PF ₆ single crystal at the cancer site was performed using a wide gap permanent magnet ESR-CT apparatus (Japanese Patent Laid-Open No. 2006-2006). It was imaged according to Example 1) of Japanese Patent No. 2455597.

  FIG. 7A (1) shows an ESR image when a micelle contrast agent with PEG is administered, and FIG. 7A (2) shows an ESR image when a DPPC micelle contrast agent is administered. These ESR images are ESR images when viewed from the back side of the cancer-bearing mouse shown in FIG. 7A (3). In the cancer-bearing mouse in FIG. 7A (3), the position of the cancer site in the left waist portion where the left and right lungs and the colon 26 are transplanted is shown. In FIG. 7A (1), since the median particle size of the micelle with PEG is as small as 0.31 μm, the state in which the contrast agent accumulates in the cancer site other than the left and right lungs and vena cava is imaged. In FIG. 7A (2), the median particle size is too large at 0.85 μm, so DPPC micelles accumulate in the left and right lungs, but not in the cancer site. In addition to the particle size, there are differences in whether the two types of contrast agents have PEG formed on the outermost surface of the micelles, but the presence or absence of accumulation at the cancer site is confirmed by imaging due to the difference in the median particle size. did it.

Furthermore, each organ including the cancer site of this mouse was taken out, and accumulation of FA ₂ PF ₆ single crystal at the cancer site was confirmed by HPLC (high performance liquid chromatography) and ESR method. FIG. 7B shows the quantitative distribution of the FA distribution amount by HPLC of micelles with PEG. The absolute amount of accumulation is higher in the heart, liver, and kidney than in the cancer site, but the ESR image is taken only in the cancer site. In the liver and kidney, it is considered that the FA ₂ PF ₆ single crystal is decomposed into FA (fluoranthene) that does not emit an ESR signal. In the heart, it is considered that the residue of the blood circulation in the form of the decomposition of the FA ₂ PF ₆ single crystal is accumulated, and that the FA ₂ PF ₆ single crystal metabolized in the liver kidney is accumulated. Seem. Despite the fact that the amount of FA per tissue weight at the cancer site is smaller than that of the heart, liver, and kidney, the reason why ESR images are taken is that cancer tissue has a special location in the cancer (large new blood vessels This is probably because the FA ₂ PF ₆ single crystals were accumulated in the place where they occurred.

[Example 2]
Next, examples in which TAM molecules (US Pat. No. 5,530,140) are used will be described. Although the TAM molecule itself is known as a carbon radical, in this example, the contrast agent of the present invention was prepared using the following two types of TAM molecules, C135 and C136. The molecular structure is shown in FIG.
C135: Tris (8-carboxy-2,2,6,6-tetramethyl-benzo (1,2-d: 4,5-d ') bis (1,3) dithiole-4-yl) methyl sodium salt,
C136: Tris (8-carboxy-2,2,6,6-tetra (2- (1-hydrokyethl))-benzo (1,2-d: 4,5-d ') bis (1,3) dithiole- 4-yl) methyl sodium salt

  A TAM stock solution was prepared by dissolving 1 g of C135 or C136 in 10 mL of physiological saline or pure water, and C135 or C136 was encapsulated in the liposomes using the drying process apparatus shown in FIG. 3A. Specifically, lecithin extracted from egg yolk by the method of Pangborn (phosphatidylcholine: Phosphatidylcoline) was dissolved in a solution of chloroform / methanol (9: 1), and the drying process apparatus shown in FIG. 3A was used. It was made to dry on the insulator flask wall surface. Next, the TAM stock solution was placed in a flask and irradiated with ultrasonic waves for 1 hour while being cooled to about 4 ° C. in an atmosphere of nitrogen gas. Thus, a milky stable liposome suspension was obtained. This suspension was centrifuged (30000 g (acceleration of gravity), 1 hour, 4 ° C.) and filtered (0.1 μm membrane filter), and then gel was added using Zephyrose 4B to obtain uniform liposomes. Filtered.

  Thus, the high concentration TAM molecule inclusion luposome contrast agent shown to FIG. 1B was created. The particle size is about 100 nm, and the mouse dynamics of the liposome-type preparation can be imaged.

[Example 3]
Next, examples in which Li-TCNQ (7,7,8,8-tetracyanoquinonedimethane) fine powder crystals are used will be described.

  Li-TCNQ crystal powder was synthesized by a diffusion method. The procedure is as follows. 2 g of commercially available 98% TCNQ was recrystallized twice with 100 mL of acetonitrile. 98% TCNQ is ocher and turns green when dissolved in acetonitrile, but becomes recrystallized into yellow scale crystals, and when dissolved in acetonitrile, becomes original yellow without impurities. When 1.2 g of recrystallized TCNQ is dissolved in 100 mL of acetonitrile and 2.121 g of lithium iodide (LiI) are dissolved in 10 mL of acetonitrile while heating, a yellow transparent liquid is obtained. When these were mixed, blackish purple Li-TCNQ fine particles were precipitated. The mixture was allowed to cool to room temperature, separated with filter paper, washed with acetonitrile, and then vacuum dried to obtain 1.096 g of 20-50 μm needle-like Li-TCNQ microcrystals. After drying, it was stored in nitrogen gas.

Next, the absorption line width ΔH and g value of the Li-TCNQ powder were evaluated by X-band ESR. The result was g = 2.0026, a value close to free electrons and FA ₂ PF ₆ , and the absorption line width ΔH was a value close to ΔH = 600 mG and the X-band ESR value of powder FA ₂ PF ₆ . The absorption line width ΔH at 200 MHz ESR was ΔH = 234 mG. The absorption line width ΔH is almost an order of magnitude larger than the absorption line width ΔH (50-60 mH) of the FA ₂ PF ₆ single crystal at the same X-band ESR. This is because the g-value anisotropy of Li-TCNQ is 5 times or more larger than that of FA ₂ PF ₆ .

Li-TCNQ fine particles (particle size 20 μm) were found to be readily soluble in water, and it was found that the surface chemical properties differed significantly from FA ₂ PF ₆ microcrystals that were not soluble in water. Therefore, it was difficult to form nanoparticles directly in water. In a solution in which a salt having a higher solubility than Li-TCNQ is dissolved, it is considered that the “salting out effect”, in which a salt having a low solubility is difficult to dissolve, can be used for making nanoparticles of Li-TCNQ. And Li-TCNQ was dissolved in an aqueous solution in which NaCl was dissolved. As a result of experiments, it was found that depending on the concentration of KCl or NaCl, the ionization of Li-TCNQ was suppressed and insolubilized, and therefore, it was applied to the nano-particulation of Li-TCNQ needle crystal powder. Specific manufacturing conditions are shown in Table 6.

  FIG. 9 shows the measurement results of the particle size distribution when the KCl concentration is fixed at 0.5 wt%, the other conditions are set to the production conditions shown in Table 6, and the pulverization time is changed. A fine particle dispersion system in an aqueous solution having a median diameter of 170 to 190 nm could be realized, and the absorption line width was about 300 mG. SEM (S-5200) observation was performed about the sample which dried the aqueous solution of FIG. 9 on Al foil. As a result, from the SEM observation, it was confirmed that nanoparticles having a size of 0.1 μm or less were aggregated.

  The process of encapsulating liposomes is almost the same as in Example 2. Lecithin (Phosphatidylcoline) extracted from egg yolk by the method of Pangborn is dissolved in a solution of chloroform / methanol (9: 1), and using a drying process apparatus (evaporator) shown in FIG. It was allowed to dry on the wall. Next, when the Li-TCNQ nanoparticle solution dispersed in the KCl solution is added to this insulator-type flask, and the mixture is cooled to about 4 ° C. in an atmosphere of nitrogen gas, it is irradiated with ultrasonic waves for 1 hour, and the emulsion color becomes stable. Liposome suspension was obtained. This suspension was centrifuged (30000 g (acceleration of gravity), 1 hour, 4 ° C.) and filtered (0.1 μm membrane filter), and then gel was added using Zephyrose 4B to obtain uniform liposomes. Filtered. In this way, a high-concentration Li-TCNQ nanoparticle-encapsulated luposome contrast agent shown in FIG. 1B was prepared. That is, the ESR contrast agent shown in FIG. 1B was produced by including the Li-TCNQ dispersion in the liposome instead of the TAM molecule aqueous solution used in forming the liposome in Example 2.

  K-TCNQ and Na-TCNQ fine powder crystals can be easily synthesized from potassium iodide (KI), sodium iodide (NaI) and acetonitrile in the same manner as Li-TCNQ. In addition, the “salting out effect” has the same effect even when K-TCNQ or Na-TCNQ fine powder crystals are used instead of Li-TCNQ. By using the process shown in the embodiment of the present invention, FIG. The indicated ESR contrast agents can be manufactured.

  In this example, the case where Li-TCNQ, K-TCNQ, and Na-TCNQ nanoparticles are dispersed in an aqueous solution has been described. However, since these TCNQ salts are water-soluble as physical properties, they are dispersed in an oily solution (non-aqueous solution). It's easy to do. Specifically, 1 g of Li-TCNQ, K-TCNQ, or Na-TCNQ fine powder crystals in 20 mL of soybean oil is mixed with an appropriate dispersant, for example, glyceryl oleate 0.15 g, and dispersed by a wet bead mill. A nano fine particle-dispersed soybean oil solution in which Li-TCNQ, K-TCNQ, and Na-TCNQ fine powder crystals were made into fine particles in soybean oil was prepared. After that, the nano-particles just prepared are dispersed in 20 mL of physiological saline, and 2 mL of soybean oil solution and 1.5 g of lecithin derived from egg yolk are administered, followed by ultra-high speed stirring at 20000 rpm using an ultra-high speed emulsifier. The structure shown can be made.

  It was possible to provide a means for imaging blood kinetics of a lipo preparation (for example, produced by LTT Laboratories) using a dispersed soybean oil solution.

[Example 4]
Next, an example in which an X-phase single crystal of LiPc (lithium phthalocyanine; C ₃₂ H ₁₆ LiN ₈ ) is used will be described. In the following description, only parts different from the first embodiment will be described. The LiPc crystal manufacturing apparatus is the crystal growth cell shown in FIG. 2A. The crystal raw material is commercially available Li ₂ Pc (Dilithium phthalocyanine), which was purified by recrystallization with ethanol.

Table 7 shows the crystal growth conditions. Since the LiPc crystal is less brittle than the FA ₂ PF ₆ single crystal, the crystal encapsulated in the phospholipid shown in FIG. 3A was obtained by the electrolytic crystal growth method like the FA ₂ PF ₆ single crystal powder. Several millimeters of crystals cannot be used as they are. Therefore, it is necessary to make fine particles about 0.1-0.9 μm. Here, the fine particles were formed into a size of about 0.1 to 0.9 μm by a wet bead mill method.

  Specific examples of conditions are shown in Table 8. By this method, LiPc microcrystals having a median diameter of 0.2 μm were obtained. After washing with cyclohexane and washing with chloroform, 0.2 μm LiPc microcrystals (in the range of 60-600 μmol) were mixed in a mixed solution of cyclohexane and chloroform and applied to the step of wrapping with phospholipid shown in FIG. 3A. This step is the same as in Example 1. Thereafter, physiological saline is placed in the flask, and after the thin film formed on the wall surface is peeled off, ultra-high speed stirring is performed with an ultra-high speed emulsifier, and a LiPc microcrystal contrast medium having a median diameter of 0.2 μm as shown in FIGS. 1A and 6A. I was able to get the agent.

The contrast agent of the present invention of LiPc can image the cancer site accumulation effect of the DDS carrier in the same cancer-bearing mouse as in Example 1, but LiPc uses the fact that the absorption line width is sensitive to the oxygen concentration in the living body. The difference from the FA ₂ PF ₆ crystal is that the oxygen concentration around the deposition site can be imaged.

[Example 5]
Next, an example using nanoparticles of YIG (Y ₃ Fe ₅ O ₁₂ : yttrium iron garnet) crystal will be described.

YIG single crystal fine powder (particle size 1-2 μm) is commercially available, and the shape is also a desirable lump shape as a starting point. For the microparticulation, the wet bead mill method schematically shown in FIG. 10 was repeated three times to form nanoparticulates. Specifically, as the first stage of micronization, water (20.5 g: 20.5 mL), YIG single crystal fine powder (particle size 1-2 μm) (20.5 g: 20.5 mL), 0.1 mmZrO ₂ ( 138 g: 37.5 mL) and a dispersant sodium polyacrylate {Aron T-40 (1.5 g: 1.5 mL)} were continuously pulverized at a rotation number of 2650 rpm for 10 hours, and pulverized into fine particle powder having a particle diameter of 100 nm. As the second stage of micronization, ZrO ₂ was replaced, water (20.5 g: 20.5 mL), YIG single crystal fine powder (particle size 1-2 μm) (20.5 g: 20.5 mL), 0.03 mm ZrO ₂ (138 g), Dispersant Aron T-40 (1.5 g: 1.5 mL) was continuously pulverized at a rotational speed of 2650 rpm for 10 hours, and pulverized into nanoparticle powder having a median diameter of 10 nm. As a third step, replace the _{ZrO 2, 0.015mmZrO 2 (138g)} , dispersing agent Aron T-40 (1.5g: 1.5mL) for 10 hours of continuous grinding at a rotation speed 2650 rpm, the median size 4nm nano Grinded into fine particle powder. Table 9 shows the YIG nanoparticle production conditions.

  The YIG nanoparticles prepared in this manner were thoroughly washed with running water, then heated and dried, and the vapor of HMDS (Hexamethyldisilazane) solution was sprayed onto the surface of the YIG nanoparticles to modify the surface of the YIG nanoparticles to be hydrophobic.

Several types of contrast agents were prepared using such YIG nanoparticle. The first is the structure of FIGS. 1A and 6A, YIG micelles. Specifically, it is the same as the method for producing micelles with phospholipids of Example 4. That is, the crystal wrapped in the phospholipid shown in FIG. 3A is YIG nanoparticle. The amount of YIG nano-particles similar to the amount of FA ₂ PF _{6 in} Table 5 is used. The method for producing the micelle is the same as in the first embodiment. However, unlike Example 1, the concentration process using a centrifuge is not used. The particle size distribution of the completed micelle YIG nanoparticulate contrast agent was a median diameter of 4.04 nm as shown in FIG. 11A.

  The second is the YIG nanoparticle contrast agent preparation step prepared above, and finally the contrast agent in the form of FIG. 1B, which is prepared using a centrifugal concentration process. That is, the micelle constituting the YIG nanoparticulate contrast agent itself can be regarded as a hydrophilic YIG nanoparticle, which is the same as effectively making the surface of the YIG nanoparticle hydrophilic. This concentrated hydrophilic YIG nanoparticulate contrast agent was encapsulated in liposomes according to Example 2 to produce a contrast agent in the form of FIG. 1B.

Third, the surface of YIG nanoparticles is hydrophilized by Ar / O ₂ plasma treatment, or ammonia water: hydrogen peroxide water: pure water = 4: 1: 6, with a liquid temperature of 60 [° C.] for 2 minutes. Thus, the contrast agent of the form shown in FIG. 1C is produced by hydrophilizing the surface of the YIG nanoparticle.

  As in Example 3, the contrast agent of the form shown in FIG. 1C was prepared, and 1 g of YIG nanoparticle hydrophilized in 20 mL of soybean oil was mixed with an appropriate dispersant, for example, 0.15 g of glyceryl oleate, and ultrahigh-speed emulsification was performed. A YIG nanoparticle-dispersed soybean oil solution was prepared by dispersing with a machine. Thereafter, 2 mL of the YIG nanoparticle-dispersed soybean oil solution just prepared in 20 mL of physiological saline and 1.5 g of lecithin derived from egg yolk are administered, and then stirred at 20000 rpm with an ultrahigh-speed emulsifier to obtain the structure shown in FIG. 1C. I was able to make.

Since YIG nanoparticle can modify the surface to be lipophilic and hydrophilic, it is advantageous in that it can constitute various contrast agents. Even if MnOFe ₂ O ₃ is used, the same fine particles can be produced. However, the particle size that can be used as an ESR contrast agent is as small as about 2 nm, and the controllability of the particle size is difficult.

  Since the YIG nanoparticulate contrast agent has very high ESR signal sensitivity, it is advantageous in that it can capture high-quality ESR images even when the accumulation effect in a minute cancer tissue and the frequency is as low as 200-300 MHz. YIG nanoparticle contrast medium accumulation was confirmed even in a colon 26 cancer strain with a tumor size of 0.3 g smaller than that of the cancer-bearing mouse of Example 1.

[Example 6]
Next, an embodiment of a contrast agent in which nanoparticles of an ESR object are embedded in a gel or jelly-like substance in a matrix will be described.

FIG. 12A shows an ESR object 101 (in this case, FA ₂ PF ₆ microcrystal, LiPc (X phase) microcrystal, Li-TCNQ microcrystal, K-TCNQ microcrystal, Na-TCNQ microcrystal in gel or jelly-like substance 91). Crystal, TAM molecular solid, etc.) are dispersed and the surface 103 is hydrophobized, whereby a structure including a surfactant such as phospholipid shown in FIG. 1A can be produced. A specific manufacturing method is disclosed below.

ESR object 101 (FA ₂ PF ₆ microcrystal LiPc (X phase) microcrystal, Li-TCNQ microcrystal, K-TCNQ microcrystal micronized to about 0.1 μm in mucus of red algae such as Tengusa (Amakusa) and Ogonori , Na-TCNQ microcrystals, TAM molecular solids, etc.), stir until uniform, freeze and dry, pulverize to about 20 μm with a dry pulverizer, hydrophobize by HMDS treatment, and into physiological saline The micelle contrast agent shown in FIG. 12A having a particle size of about 1 μm was prepared by ultrahigh-speed emulsification together with the phospholipids shown in Table 5.

In this method, FA ₂ PF ₆ microcrystals, LiPc (X phase) microcrystals, Li-TCNQ microcrystals, K-TCNQ microcrystals, Na-TCNQ microcrystals, TAM molecular solids, etc. are dispersed and modified in collagen. It is also possible to prepare gelatin by adding gelatinizer to gelatin to prepare a micelle contrast agent shown in FIG. 12A.

  Further, as shown in FIG. 12B, an ESR object 51 was dispersed in a gel or jelly-like substance 41 using an AB type block copolymer consisting of hydrophilic polyethylene glycol 22 and hydrophobic polyamino acid 32 as a DDS carrier. A micelle structure can also be produced.

  In the present invention, an ESR contrast agent can be produced by including various types of ESR objects depending on the types of known DDS carriers.

  In this contrast agent, it was found that DDS carrier adsorbed unexpectedly to the vena cava of the mouse, whether it was adsorbed as DDS carrier, or the contrast agent embedded in the matrix form in which DDS carrier was broken was exposed to the outside However, it was found that the DDS carrier was broken and a contrast agent embedded in a matrix was deposited. As can be seen from this example, it has a role different from conventional contrast agents in that the unexpected behavior of the DDS carrier can be understood. He showed me the opportunity to review how to make a DDS carrier (including how to wrap ESR contrast media).

[Example 7]
FIG. 13 shows an example of a DDS carrier that is screened for cancer formed in the intestinal wall. The contrast agent is an oral contrast agent. As the structure, FIG. 13A has almost the same structure as FIG. 1A, and FIG. 13B has almost the same structure as FIG. 1B, and can be manufactured by the method described in Example 1-6. However, the particle size is as large as 50 μm to 100 μm, respectively. FA ₂ PF ₆ crystal, LiPc (X phase) crystal, Li-TCNQ microcrystal, K-TCNQ microcrystal, Na-TCNQ microcrystal, The process of refining the TAM molecule solution is greatly simplified. YIG's nanoparticulation has not changed. Further, in order to selectively adhere to cancer existing in the intestinal wall, the point that the antibody 39 that is good for binding to the cancer site is formed on the surface of the DDS carrier is different from the structure of FIG. 1A or 1B. .

  As an example for other gastric cancers, in the case of the immunoliposome preparation MCC465 in which an antibody and liposome are modified with human antibodies and the anticancer drug doxorubicin is sealed, has the immunoliposome preparation reached the desired cancer tissue? Alternatively, in order to observe whether an unintended tissue has been reached, the dynamics of the DDS carrier can be imaged by making a contrast agent having a structure of FIG. 13B with a particle size of about 200 nm. In this case, the DDS carrier is formed identical to the immunoliposome formulation MCC465.

[Example 8]
If this invention is used, the distribution of DDS can be imaged by mixing and using with a DDS pharmaceutical. For example, by incorporating the anticancer drug doxorubicin hydrochloride into the liposome of Example 2, the DDS carrier having the same structure as the ESR contrast agent can be included in the liposome of Example 2 to know the in vivo dynamics. it can. In another example, prostaglandin E1 (PGE1), which is a lipo preparation, can be dissolved in the oily solution 9 of FIG. 1C as a lipophilic pharmaceutical compound to simultaneously produce the pharmaceutical and the ESR contrast agent. In this case, the pharmacokinetics until reaching the tissue of interest can be known from the in vivo kinetics of the ESR contrast agent. About the cerebral action after the administration, a certain amount of information can be obtained by imaging the disintegration process of the contrast agent in the living body.

The schematic diagram which shows the basic structure of the contrast agent for ESR by this invention. The schematic diagram which shows the basic structure of the contrast agent for ESR by this invention. The schematic diagram which shows the basic structure of the contrast agent for ESR by this invention. The figure which showed the relationship between the median particle diameter d when a superparamagnetic YIG nanoparticle is disperse | distributed in the solution of viscosity coefficient (eta), and ESR absorption line width (DELTA) H in a low magnetic field. Explanatory drawing of the crystal growth cell used by this invention. Explanatory drawing of the conventional crystal growth cell. The figure which shows the example of the cathode container with a filter. The figure which shows the example of the cathode container with a filter. The schematic diagram of the apparatus which produces the ESR contrast agent of this invention. Explanatory drawing of the process of producing the ESR contrast agent of this invention. It shows a specific example of FA ₂ PF ₆ single crystallization yield obtained by the crystal growth cell of the present invention. The particle size distribution map of DPPC micelles. Structure diagram of micelle with PEG. It shows an example of the particle size distribution of FA ₂ PF ₆ crystalline micelle contrast agent. Graph showing measured values of the FA ₂ PF ₆ single crystal concentration in the contrast medium. Shows the ESR-CT images when administered to PEG with FA ₂ PF ₆ monocrystalline micelles contrast medium into tumor-bearing mice. The figure which shows FA distribution to each organ obtained by HPLC (high performance liquid chromatography) analysis. Molecular structure diagram of TAM molecule. The particle size distribution figure of Li-TCNQ nanoparticle. The conceptual diagram of a wet bead mill, and explanatory drawing of an operation principle. The figure which shows the example of the particle size distribution of a YIG nanoparticle contrast agent. The figure which shows the example of the ESR contrast agent of this invention. The figure which shows the example of the ESR contrast agent of this invention. The figure which shows the example of the ESR contrast agent of this invention. The figure which shows the example of the ESR contrast agent of this invention.

Explanation of symbols

1, 8, 51, 101 ... ESR object, 2, 4, 21, 21 '... hydrophilic group, 3, 31, 32, 31' ... lipophilic group (hydrophobic group), 6, 22 ... PEG, 7 ... aqueous solution, DESCRIPTION OF SYMBOLS 9 ... Non-aqueous solution, 41, 91 ... Gel or jelly-like substance, 11 ... Surfactant, 103 ... Hydrophobized surface, 32 ... Polyamino acid, 39 ... Antibody, 60 ... Cylindrical container, 61 ... Cathode container with cylindrical filter 62 ... Common cathode, 63 ... Teflon lid, 64 ... Vacuum drawing window, 65, 72 ... Anode, 66 ... Filter, 67 ... Anode region, 68, 75 ... Filter, 71 ... Erlenmeyer flask type anode region, 73 ... Cylindrical shape Cathode region, 74 ... cathode, 76 ... vacuum cock

Claims (20)

  A contrast agent for ESR, wherein a single or a plurality of ESR objects exhibiting electron spin resonance (ESR) are included in a structure whose outermost surface is composed of a hydrophilic group.   2. The contrast agent for ESR according to claim 1, wherein the electron spin resonance absorption line width ΔH is 1000 m gauss or less under a static magnetic field of 50 to 500 gauss.   3. The contrast agent for ESR according to claim 2, wherein the structure has a median particle diameter of 1000 μm or less. In ESR imaging agent of claim 2, wherein, ESR imaging agent, wherein the ESR object is composed of FA ₂ PF ₆ crystal, or Li-Pc crystals. 3. The contrast agent for ESR according to claim 2, wherein the ESR object is composed of a single FA ₂ PF ₆ crystal or a single X-phase Li—Pc crystal, and the structure has a lipophilic group on the crystal surface. A contrast agent for ESR, which is formed by a surfactant that is formed and has a hydrophilic group exposed on the surface side.   The contrast agent for ESR according to claim 2, wherein the ESR object is a superparamagnetic YIG crystal, and a crystal median particle size is 10 nm or less.   7. The contrast agent for ESR according to claim 6, wherein the superparamagnetic YIG crystal has a median minimum length to maximum length ratio (maximum length / minimum length) of 1.5 or less.   7. The contrast agent for ESR according to claim 6, wherein the structure is formed of a surfactant in which a lipophilic group is adsorbed on the surface of the superparamagnetic material YIG and a hydrophilic group is exposed on the surface side. Contrast agent for ESR.   7. The contrast agent for ESR according to claim 6, wherein the superparamagnetic YIG crystal is included by a surfactant whose surface is hydrophilized or whose lipophilic groups are adsorbed on the crystal surface and hydrophilic groups are exposed on the surface side. The structure is dispersed in an aqueous solvent, the structure has a liposome structure, and includes a plurality of the superparamagnetic YIG crystals dispersed in the aqueous solvent. Contrast agent.   7. The contrast agent for ESR according to claim 6, wherein the superparamagnetic YIG crystal has a hydrophobic surface and is dispersed in an oily solvent.   11. The contrast agent for ESR according to claim 10, wherein the structure is formed of lecithin and includes therein a plurality of the superparamagnetic YIG crystals dispersed in the oily solvent. Contrast agent.   The contrast agent for ESR according to claim 2, wherein the outermost surface of the structure is formed of a phospholipid containing a phospholipid with polyethylene glycol.   The contrast agent for ESR according to claim 12, wherein the phospholipid is L-αdipalmitoylphosphatidylcholine or L-αdistearoylphosphatidylethanolamine.   3. The contrast agent for ESR according to claim 2, wherein the structure is made of a non-solution substance, and the ESR object is dispersed in the non-solution substance.   The contrast agent for ESR according to claim 2, wherein the ESR object is composed of Li-TCNQ, K-TCNQ, Na-TCNQ, or an aqueous TAM molecule solution.   The contrast agent for ESR according to claim 2, wherein the median diameter of the structure is 400 nm or less.   3. The contrast agent for ESR according to claim 2, wherein the contrast agent for ESR has a structure in which the structure is included in another structure having a larger size. 5. The contrast medium for ESR according to claim 4, wherein the FA ₂ PF ₆ crystal or LiPc crystal uses a vertical crystal growth apparatus in which a plurality of anodes are arranged on the circumference with respect to a common cathode separated by a filter. A contrast agent for ESR, which is produced by the conventional electrolytic crystal growth method. A method for producing a contrast agent for ESR in which a single or a plurality of ESR objects exhibiting electron spin resonance (ESR) are included in a structure whose outermost surface is composed of a hydrophilic group,
Forming a first thin film on a container wall from a solution containing a substance constituting the structure;
Forming a second thin film on the dry-formed first thin film from a solution containing the substance constituting the structure and the ESR object;
A method of producing a contrast agent for ESR, comprising: forming a third thin film on the second thin film from a solution containing a substance constituting the structure. Single or multiple ESR objects exhibiting electron spin resonance (ESR) are included in a first structure whose outermost surface is composed of a hydrophilic group, and electron spin is performed under a static magnetic field of 50 to 500 gauss. A contrast agent for ESR having a resonance absorption line width ΔH of 1000 m gauss or less;
A contrast agent, wherein a DDS drug containing a drug is mixed in a second structure having the same chemical structure as that of the first structure on the outermost surface.

Images