JP2012097067A - Contrast medium for optoacoustic imaging and optoacoustic imaging method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a contrast medium for optoacoustic imaging that generates a large optoacoustic signal.SOLUTION: The contrast medium for optoacoustic imaging contains iron-oxide particles whose particle size is 15 nm or more and 500 nm or less.

Description

  The present invention relates to a contrast agent for photoacoustic imaging and a photoacoustic imaging method using the same.

  A photoacoustic imaging method for visualizing information inside a living body is known. The photoacoustic imaging method is a method of obtaining an image of substance distribution inside a specimen by measuring the intensity and generation position of a photoacoustic signal emitted from the specimen by irradiating the specimen with light.

  Here, it is known that when Rhizovist (registered trademark) (contrast agent for MRI in which a plurality of iron oxide particles are coated with a polysaccharide dextran) is irradiated with light, an acoustic wave is generated (non-patent document). 1). Therefore, there is a possibility that Resovist (registered trademark) can be used as a contrast agent for photoacoustic imaging.

Biomed Tech 2009; 54: pp. 83-88

  However, the iron oxide particles constituting Resovist (registered trademark) have a small particle size, which is 5 nm according to the measurement by the inventors. When the particle diameter of the iron oxide particles is small, the generated acoustic wave is small and the photoacoustic signal to be measured is small. Accordingly, an object of the present invention is to provide a contrast agent for photoacoustic imaging in which a large acoustic wave is generated and a photoacoustic signal to be measured is large.

  The contrast agent for photoacoustic imaging according to the present invention is characterized in that, in the contrast agent for photoacoustic imaging having iron oxide particles, the iron oxide particles have a particle size of 15 nm to 500 nm.

  According to the present invention, since the iron oxide particles that emit a strong photoacoustic signal are used, it is possible to provide a contrast agent that emits a photoacoustic signal superior to that of the prior art.

It is the schematic which shows the structure of the contrast agent for photoacoustic imaging which concerns on embodiment of this invention. It is the schematic which shows the structure of the contrast agent for photoacoustic imaging which concerns on embodiment of this invention. It is a figure which shows an example of the process of manufacturing the contrast agent for photoacoustic imaging which concerns on embodiment of this invention. It is a waveform of photoacoustic signal intensity. It is a figure which shows the relationship between the particle size of an iron oxide particle, a molar absorption coefficient, and a photoacoustic signal. It is an evaluation result of contrast agent 1 for photoacoustic imaging (NP1). It is an evaluation result of contrast agent 2 for photoacoustic imaging (NP2). It is a figure which shows stability of the contrast agent 2 (NP2) for photoacoustic imaging. (A) It is a cryo-TEM image of contrast agent 3 for photoacoustic imaging (NP3). (B) Cryo-FIB / SEM image of contrast agent 3 for photoacoustic imaging (NP3). It is a cryo-TEM image of contrast agent 6 (NP6) for photoacoustic imaging. It is a TEM image of contrast agent 7 (NP7) for photoacoustic imaging. It is a figure which shows stability of contrast agent 12 for photoacoustic imaging (NP12). It is a figure which shows the cancer accumulation property of the contrast agent 12 for photoacoustic imaging (NP12). It is a figure which shows the relationship between the particle size of the contrast agent for photoacoustic imaging, and photoacoustic signal strength.

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

  A contrast agent for photoacoustic imaging according to the present embodiment (hereinafter sometimes abbreviated as a contrast agent) has iron oxide particles, and the particle size of the iron oxide particles is 15 nm to 500 nm. When the particle diameter of the iron oxide particles is 15 nm or more, the value of the molar extinction coefficient per mole of iron atoms and the value of the photoacoustic signal measured from one mole of iron atoms are larger than when the particle diameter is less than 15 nm. . Therefore, a large photoacoustic signal can be obtained by using the contrast agent according to the present embodiment in the photoacoustic imaging method. Further, the particle diameter of the iron oxide particles is more preferably 20 nm or more, and further preferably 50 nm or more. Further, the iron oxide particles preferably have a particle size of 100 nm or less.

  The particle size of the contrast agent according to this embodiment is preferably 15 nm or more and 1000 nm or less. When the particle size of the contrast agent is 1000 nm or less, more contrast agent can be accumulated in the tumor site than in the normal site in the living body due to the EPR (Enhanced Permeability and Retention) effect. As a result, when the contrast medium is administered into the living body and then the living body is irradiated with light, the photoacoustic signal emitted from the tumor site becomes larger than the photoacoustic signal emitted from the normal site. Therefore, the tumor site can be specifically detected by setting the particle size of the contrast agent to 1000 nm or less. Further, the particle diameter of the contrast agent is more preferably 500 nm or less, and further preferably 200 nm or less. This is because when the particle size of the contrast agent is 200 nm or less, the contrast agent is less likely to be taken up by macrophages in the blood, and the retention in blood is considered to be high.

  In this specification, the particle diameter of the contrast agent is a fluid dynamics measured by a dynamic light scattering (DLS) method using a dynamic light scattering analyzer (DLS-8000, manufactured by Otsuka Electronics Co., Ltd.). It means the target diameter. For the particle size, 100 times of integration is performed 5 times using the above apparatus, and an average of the calculated 5 data is taken.

  The contrast agent according to the present embodiment may have only one iron oxide particle or two or more. When the particle diameter of the iron oxide particles contained in the contrast agent is 500 nm, the number of iron oxide particles contained in the contrast agent is preferably 3 or less. In addition, when the particle diameter of the iron oxide particles is large, for example, in the case of 50 nm, as the number of iron oxide particles contained in the contrast agent increases, the value of the molar extinction coefficient per mole of iron atoms and The value of the photoacoustic signal measured from is large. On the other hand, when the particle diameter of the iron oxide particles is 5 nm, even if the number of iron oxide particles contained in the contrast agent increases, the molar extinction coefficient per mole of iron atoms and the value of the photoacoustic signal to be measured are It does n’t change much.

  The iron oxide particles included in the contrast agent according to this embodiment may be primary particles or secondary particles.

  In this embodiment, the surface of the primary particle of the iron oxide particle or the surface of the secondary particle may be coated with a surface modifier. Examples of the surface modifier include fatty acids and amphiphilic compounds. Alternatively, an amphiphilic compound may be bound to a hydrophobic polymer containing iron oxide particles or iron oxide particles coated with a fatty acid. Examples of amphiphilic compounds include phospholipids, polyoxyethylene sorbitan fatty acid esters, and amphiphilic polymers. In the present embodiment, one type of surface modifier may be used, or a plurality of types may be used.

(Example of contrast agent for photoacoustic imaging)
A schematic diagram of an example of a contrast agent for photoacoustic imaging (hereinafter sometimes abbreviated as a contrast agent) according to the present embodiment is shown in FIG. The contrast agent 1 for photoacoustic imaging which concerns on this embodiment consists of the iron oxide particle 2 and the amphiphilic compound 3 provided as needed as shown to Fig.1 (a). The amphiphilic compound 3 is present on the surface of the contrast agent 1 for photoacoustic imaging.

  When the iron oxide particles 2 are irradiated with light, the iron oxide particles 2 generate an acoustic wave and detect it as a photoacoustic signal. In addition, the presence of the amphiphilic compound on the surface of the contrast agent 1 for photoacoustic imaging can suppress aggregation of the contrast agent for photoacoustic imaging in water or an aqueous solution.

(Iron oxide particles)
In the present invention, the iron oxide particles have a particle size of 15 nm to 500 nm.

The iron oxide particles in the present embodiment are not particularly limited, but have absorption in the near infrared wavelength region of 600 to 1300 nm. Specific examples of the iron oxide particles include Fe ₃ O ₄ (magnetite), γ-Fe ₂ O ₃ (maghemite), and a mixture thereof. Particularly preferred is magnetite. Magnetite is known to have a higher molar extinction coefficient in the near-infrared wavelength region than maghemite. Therefore, it is considered that the acoustic wave generated when irradiated with light is also increased.

  Further, the iron oxide particles in the present embodiment may be in any single crystal, polycrystal, or amorphous state.

As the iron oxide particles in the present embodiment, commercially available particles may be used, or those obtained by the following method may be used. For example, FeCl ₃ and FeCl ₂ are dissolved in water to form a solution, and ammonia water is added while stirring the solution to obtain iron oxide particles.

(Iron oxide particle size)
In this embodiment, the particle size of iron oxide particles is the particle size of primary particles of iron oxide measured from an image obtained using, for example, a transmission electron microscope (TEM). When the shape of the iron oxide particles was not spherical, the lengths of the short axis and long axis of the image of the iron oxide particles were measured, and the average of these values was taken as the diameter.

(fatty acid)
In this embodiment, examples of the fatty acid include oleic acid. Iron oxide particles coated with oleic acid are obtained as follows. First, an organic solvent is added to the dried iron oxide particles to form a dispersion solution of iron oxide particles, and oleic acid is added to the dispersion solution to coat the surfaces of the iron oxide particles with oleic acid. Thereafter, oleic acid that does not exist on the surface of the iron oxide particles is washed with an organic solvent to obtain iron oxide particles coated with oleic acid. Note that the compound is not limited to oleic acid as long as it is a compound that can bind or adsorb an amphiphilic compound or can disperse iron oxide particles in an organic solvent contained in the first liquid described later.

(Amphiphilic compounds)
In the present embodiment, the amphiphilic compound is preferably at least one selected from phospholipids, polyoxyethylene sorbitan fatty acid esters, and amphiphilic polymers.

(Phospholipid)
In the present embodiment, the phospholipid may be any lipid as long as it has a phosphate. The phospholipid in the present embodiment preferably includes a phosphatidyl phospholipid having polyethylene glycol (Polyethylene Glycol, hereinafter abbreviated as PEG). When the iron oxide particles according to the present embodiment have a phospholipid having PEG, it is considered that there is an effect of suppressing the adsorption of the contrast agent to the protein by increasing the volume of the contrast agent by collecting water molecules in the PEG. It is done. Moreover, when PEG is contained, it is difficult to be phagocytosed by macrophages. Moreover, when it has PEG, it is hard to aggregate in water or aqueous solution.

  In addition, when binding a capture molecule such as an antibody, which will be described later, to a phospholipid, it is preferable to bind the capture molecule to a phospholipid having an amino group, a carboxyl group, an NHS group, or a maleimide group via these functional groups. .

As the phospholipid according to the present embodiment, for example, N- (Aminopropoxypolyethyleneglycol) carboyl-disteaylylphosphatidyl-ethanolamine (DSPE-PEG-NH ₂ ) represented by Chemical Formula 1 and 3- (N-Glutopylpropyl) represented by Chemical Formula 2 -Carbonyl disteayl phosphatidyl-ethanolamine (DSPE-PEG-COOH), 3- (N-Succinimidyloxyglutaryl) aminopropylidyl-polyethylidyl-glycolyl-carbonyl-carboxylic acid hanolamine (DSPE-PEG-NHS), N-[(3-Maleimide-1-oxopropyl) aminopropyleneglycolyl-carboxylic-carbon-DSN-PEG Carbonyl-methoxypolyethyleneglycol-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt (DSPE-PEG-CN), [P ly (ethylene glycol)] can be cited phospholipids (DSPE-PEG) and the like.

(Polyoxyethylene sorbitan fatty acid ester)
The polyoxyethylene sorbitan fatty acid ester in the present embodiment is not particularly limited, but is at least one polyoxyethylene sorbitan fatty acid ester selected from Tween 20 (polyoxyethylene sorbitan monolaurate), Tween 40, Tween 60, and Tween 80. It is preferable.

(Amphiphilic polymer)
As an amphiphilic polymer in this embodiment, an octadecene-maleic anhydride alternating copolymer to which PEG is bonded, a styrene-maleic anhydride copolymer to which PEG is bonded, polylactic acid to which PEG is bonded, and PEG are bonded. Examples thereof include a lactic acid-glycolic acid copolymer, polyoxyethylene polyoxypropylene, and polyvinyl alcohol. Here, the weight average molecular weight of PEG is 1000 to 100,000, preferably 2000 to 20000. Moreover, at the terminal of PEG, it has any functional group of a hydroxyl group, a methoxy group, an amino group, a carboxyl group, an NHS group, or a maleimide group, and the functional group is an amino group, a carboxyl group, an NHS group, or a maleimide group. In this case, a capture molecule can be bound.

(Hydrophobic polymer)
The hydrophobic polymer in the present embodiment may be any polymer as long as it is hydrophobic and has a function as a binder containing a plurality of iron oxide particles. Since the polymer is hydrophobic, the iron oxide particles contained therein or the iron oxide particles coated with a fatty acid hardly flow out.

  Examples of the hydrophobic polymer in the present embodiment include a homopolymer composed of a monomer having a hydroxycarboxylic acid having 6 or less carbon atoms, a copolymer composed of two or more kinds of the monomers, and a polymer having a vinyl group. The polymer having a vinyl group may be a homopolymer or a copolymer. The iron oxide particles in this embodiment are preferably coated with at least one of them. When the contrast agent according to this embodiment is administered in vivo, a polymer having a hydroxycarboxylic acid having 6 or less carbon atoms as a monomer is used as the hydrophobic polymer so that the contrast agent does not remain in the living body for a long time. Is preferred. This is because a polymer having a hydroxycarboxylic acid having 6 or less carbon atoms as a monomer has an ester bond that is cleaved by hydrolysis. Monomers whose ester bonds have been cleaved are easily metabolized, and are therefore unlikely to remain in the living body.

  Examples of the polymer having a hydroxycarboxylic acid having 6 or less carbon atoms include polylactic acid and lactic acid-glycolic acid copolymer, and examples of the polymer having a vinyl group include polystyrene and polymethyl methacrylate. The hydrophobic polymer preferably has a weight average molecular weight of 2,000 to 1,000,000, and more preferably 10,000 to 600,000.

(Other examples of contrast agents for photoacoustic imaging)
The contrast agent for photoacoustic imaging other than said example is demonstrated.
FIG. 1B is a schematic view of a contrast agent 1 for photoacoustic imaging in which iron oxide particles 2 are coated only with phospholipid 4.

  FIG. 1C is a schematic view of the contrast agent 1 for photoacoustic imaging in which the iron oxide particles 2 are coated only with the amphiphilic polymer 6.

  FIG. 1 (d) is a schematic view of a contrast agent 1 for photoacoustic imaging in which iron oxide particles 2 are coated only with an amphiphilic polymer 6, and the iron oxide particles 2 are localized on the surface of the contrast agent 1 for photoacoustic imaging. It is in existence.

  FIG. 1 (e) is a schematic view of a contrast agent 1 for photoacoustic imaging in which iron oxide particles 2 contained in a hydrophobic polymer 7 are covered only with a polyoxyethylene sorbitan fatty acid ester 5.

  FIG. 1 (f) is a schematic view of a contrast agent 1 for photoacoustic imaging in which iron oxide particles 2 contained in a hydrophobic polymer 7 are coated with both a phospholipid 4 and a polyoxyethylene sorbitan fatty acid ester 5. is there.

  FIG. 1 (g) shows a contrast agent 1 for photoacoustic imaging in which iron oxide particles 2 contained in a hydrophobic polymer 7 are coated with both a phospholipid 4 and a polyoxyethylene sorbitan fatty acid ester 5. The iron particles 2 are schematic views localized on the surface of the contrast agent 1 for photoacoustic imaging.

  FIG. 2A is a schematic view of the contrast agent 1 for photoacoustic imaging in which the iron oxide particles 2 contained in the hydrophobic polymer 7 are covered only with the amphiphilic polymer 6.

  In the contrast agent 1 for photoacoustic imaging having the phospholipid 4, a capture molecule 9 that binds to a target tissue such as a tumor, a cell, a substance, or the like can be bound to at least a part of the phospholipid 4. 2 (b), 2 (e), and 2 (f) are schematic views of a contrast agent 10 for photoacoustic imaging in which a capture molecule 9 is bound to a contrast agent 1 for photoacoustic imaging having a phospholipid 4. FIG. The contrast agent 10 for photoacoustic imaging thus obtained can also specifically bind to target tissues, cells, substances, and the like.

  In addition, in the contrast agent 1 for photoacoustic imaging having the amphiphilic polymer 6, the capture molecule 9 can be bonded to at least a part of the amphiphilic polymer 6. 2 (c), FIG. 2 (d), and FIG. 2 (g) show a contrast agent 10 for photoacoustic imaging in which a capture molecule 9 is bound to a contrast agent for photoacoustic imaging having an amphiphilic polymer 6. FIG. The contrast agent 10 for photoacoustic imaging thus obtained can be specifically bound to a target tissue, cell, substance, and the like.

  Further, the contrast agents 1 and 10 for photoacoustic imaging according to the present embodiment can control the particle size of the contrast agent for photoacoustic imaging for a target application.

(Method for producing contrast agent for photoacoustic imaging)
The manufacturing method of the contrast agent for photoacoustic imaging in this embodiment is demonstrated.

  Examples of a method for obtaining a contrast agent for photoacoustic imaging include, but are not limited to, the following dry-up method and nanoemulsion method.

An example of a process for producing a contrast agent for photoacoustic imaging by the dry-up method is shown in FIG. Specifically, an aqueous dispersion of a contrast agent for photoacoustic imaging can be obtained through the following steps (1) to (2).
(1) A step of distilling off the organic solvent from the first liquid 11 obtained by adding the iron oxide particles 2 and the phospholipid 4 or the amphiphilic polymer 6 to the organic solvent.
(2) A step of emulsifying the residue obtained in (1) by adding water 14 and then irradiating with ultrasonic waves.

An example of a process for producing a contrast agent for photoacoustic imaging by the nanoemulsion method is shown in FIG. Specifically, an aqueous dispersion of a contrast agent for photoacoustic imaging can be obtained through the following steps (1) to (3).
(1) A second liquid 11 in which iron oxide particles 2 and a hydrophobic polymer 7 are added to an organic solvent, and a second liquid in which polyoxyethylene sorbitan fatty acid ester 5, phospholipid 4 or amphiphilic polymer 6 is added to water. A step of obtaining a mixed liquid in addition to the liquid 12.
(2) A step of obtaining an O / W type emulsion 13 by irradiating the mixed solution with ultrasonic waves to emulsify.
(3) A step of distilling off the organic solvent from the dispersoid of the emulsion 13.

(First liquid)
The first liquid in the dry-up method is a liquid in which iron oxide particles and phospholipids or amphiphilic polymers are dispersed and dissolved in an organic solvent. The first liquid in the nanoemulsion method is a liquid in which iron oxide particles and a hydrophobic polymer are dispersed and dissolved in an organic solvent.

  The organic solvent contained in the first liquid can be any solvent as long as it dissolves the phospholipid or hydrophobic polymer and can disperse the iron oxide particles, but it must be a volatile organic solvent. Is preferred.

  The following solvent is mentioned as a specific example of such an organic solvent. Halogenated hydrocarbons (dichloromethane, chloroform, chloroethane, dichloroethane, trichloroethane, carbon tetrachloride, etc.), ethers (ethyl ether, isobutyl ether, butanol, etc.), esters (ethyl acetate, butyl acetate, etc.), aromatic hydrocarbons ( Benzene, toluene, xylene, etc.). These may be used alone, or two or more kinds may be mixed and used at an appropriate ratio. However, the organic solvent contained in the first liquid is not limited to the above.

  In addition, the concentration of the phospholipid, amphiphilic polymer, or hydrophobic polymer in the first liquid is not particularly limited as long as they are in a range where they can be dissolved, but a preferable concentration is, for example, 0.1 to 100 mg for phospholipid. / Ml, amphiphilic polymer 1-100 mg / ml, and hydrophobic polymer 7 0.5-100 mg / ml.

  The weight ratio of the phospholipid and iron oxide particles contained in the first liquid in the dry-up method is preferably in the range of 10: 1 to 1:10. The weight ratio of the amphiphilic polymer and the iron oxide particles contained in the first liquid in the dry-up method is preferably in the range of 10: 1 to 1: 100. The weight ratio of the hydrophobic polymer and iron oxide particles contained in the first liquid in the nanoemulsion method is preferably in the range of 1000: 1 to 1: 9.

(Second liquid)
The second liquid in the nanoemulsion method is a phospholipid and polyoxyethylene sorbitan fatty acid ester in water, each of which is a single compound, or each of them for the purpose of stabilizing the emulsion when mixed with the first liquid. It is an aqueous solution to which a plurality of compounds or amphiphilic polymers are added in advance. However, the method is not limited to the above method as long as a phospholipid, a polyoxyethylene sorbitan fatty acid ester, or an amphiphilic polymer can be contained in a dispersion obtained by mixing the first liquid and water.

  Further, the concentration of the phospholipid and polyoxyethylene sorbitan fatty acid ester contained in the second liquid in the nanoemulsion method depends on the mixing ratio of the first liquid, but as a preferable concentration, for example, as a preferable concentration of phospholipid, It can be 0.001 mg / ml to 100 mg / ml. Further, the polyoxyethylene sorbitan fatty acid ester may be 0.1 mg / ml to 100 mg / ml. The amphiphilic polymer may be 1 mg / ml to 100 mg / ml.

(Emulsification)
In the dry-up method, after a mixed solution of iron oxide particles and a phospholipid or an amphiphilic polymer is dried, a solution to which water has been added is emulsified to obtain micelles. In the nanoemulsion method, a mixed liquid of a first liquid and a second liquid is emulsified to obtain an oil-in-water (O / W) type emulsion.

  Emulsions and micelles include emulsions and micelles having any physical properties as long as the object of the present invention can be achieved, but are preferably emulsions and micelles having a single particle size distribution.

  Such emulsions and micelles can be prepared by a conventionally known emulsification technique. Conventionally known methods include, for example, an intermittent shaking method, a stirring method using a mixer such as a propeller-type stirrer, a turbine-type stirrer, a colloid mill method, a homogenizer method, and an ultrasonic irradiation method. These methods can be used alone or in combination. Further, the emulsion and micelle may be prepared by one-stage emulsification, or may be prepared by multi-stage emulsification. However, the emulsification technique is not limited to the above technique as long as the object of the present invention can be achieved.

  In particular, the emulsion in the nanoemulsion method is an oil-in-water (O / W) type emulsion prepared from a mixture of a first liquid and a second liquid. Here, the mixing of the first liquid and the second liquid means that the first liquid and the second liquid are brought into contact with each other without being spatially separated, and need not necessarily be mixed with each other.

  The ratio of the first liquid and the second liquid in the mixed liquid is not particularly limited as long as an oil-in-water (O / W) type emulsion 12 can be formed, but preferably the first liquid and the second liquid. It is preferable that the weight ratio is 1: 2 to 1: 1000.

(Distilled)
Distillation is an operation of removing the organic solvent contained in the first liquid.

  The distillation can be carried out by any conventionally known method, and examples thereof include a method of removing by heating or a method of using a decompression device such as an evaporator. The heating temperature in the case of removal by heating is preferably in the range of 0 ° C to 80 ° C. However, the distillation is not limited to the above method as long as the object of the present invention can be achieved.

(Capture molecule)
In the present invention, the capture molecule is a substance that specifically binds to a target site such as cancer, a substance that specifically binds to a substance that exists around the target site, or a chemical substance such as a biomolecule or a pharmaceutical. It is a substance selected arbitrarily. Specific examples include antibodies, antibody fragments, enzymes, biologically active peptides, glycopeptides, sugar chains, lipids, molecular recognition compounds, and the like. In the present embodiment, the capture molecule is preferably a single chain antibody.

  In the present embodiment, the capture molecule is chemically bound to a contrast agent for photoacoustic imaging having a phospholipid or an amphiphilic polymer to constitute the contrast agent for photoacoustic imaging according to the present invention. By using such a contrast agent for photoacoustic imaging, it is possible to perform specific detection of the target site and tracking of the dynamics, localization, drug efficacy, metabolism, and the like of the target substance.

(Immobilization of capture molecules)
The contrast agent for photoacoustic imaging of this embodiment can be obtained by binding a capture molecule to the contrast agent for photoacoustic imaging having a phospholipid.

  In this embodiment, as a method of binding a capture molecule to a contrast agent for photoacoustic imaging having a phospholipid or an amphiphilic polymer, the functional group of the phospholipid or the amphiphilic polymer is reacted with the functional group of the capture molecule 8. And chemical bonding methods can be used.

  That is, when the functional group of the phospholipid or amphiphilic polymer is an N-hydroxysuccinimide group, the capture molecule can be bound to the contrast agent for photoacoustic imaging by reacting with the amino group of the capture molecule. After immobilization of the capture molecule, the unreacted N-hydroxysuccinimide group of the phospholipid or amphiphilic polymer is reacted with glycine, ethanolamine, oligoethylene glycol having an amino group at the terminal, polyethylene glycol, or the like to succinimide It is preferred to deactivate the group.

  Alternatively, when the functional group of the phospholipid or amphiphilic polymer is a maleimide group, the capture molecule can be bound to the contrast agent for photoacoustic imaging by reacting with the thiol group of the capture molecule. After immobilizing the capture molecule, the unreacted maleimide group of the phospholipid or amphiphilic polymer is reacted with L-cysteine, mercaptoethanol, oligoethylene glycol or polyethylene glycol having a thiol group at the terminal, and the maleimide group Is preferably deactivated.

  Alternatively, when the functional group of the phospholipid or amphiphilic polymer is an amino group, it can be reacted with the amino group of the capture molecule 8 using glutaraldehyde to bind the capture molecule to the contrast agent for photoacoustic imaging. After binding the capture molecule, it is preferable to block the activity of the aldehyde group by reacting with ethanolamine, oligoethylene glycol having an amino group at the terminal, polyethylene glycol or the like.

  Alternatively, the amino group may be substituted with an N-hydroxysuccinimide group or a maleimide group to bind the capture molecule.

(Dispersion medium)
The contrast agent for photoacoustic imaging according to the present embodiment may further have a dispersion medium. The dispersion medium is a liquid substance for dispersing the iron oxide particles according to this embodiment, and examples thereof include physiological saline and distilled water for injection. In the contrast agent according to the present embodiment, the iron oxide particles according to the present embodiment may be dispersed in advance in the dispersion medium, or the iron oxide particles and the dispersion medium according to the present embodiment may be used as a kit. The particles may be dispersed in a dispersion medium before being administered into a living body.

(Contrast of contrast agent for photoacoustic imaging)
The contrast agent for photoacoustic imaging can be used by being dispersed in a solvent such as physiological saline or distilled water for injection. Moreover, you may add a pharmacologically acceptable additive suitably as needed. These contrast agents for photoacoustic imaging can be introduced into the body by intravenous injection or subcutaneous injection.

(Photoacoustic imaging method)
The photoacoustic imaging method according to the present embodiment includes the following steps.
(I) a step of irradiating the sample to which the contrast agent for photoacoustic imaging is administered with light in a wavelength region of 600 nm to 1300 nm (ii) a step of detecting an acoustic wave generated from the contrast agent present in the sample Here, an apparatus for detecting acoustic waves is not particularly limited, but an ultrasonic probe or the like can be used.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and light having similar functions and effects such as materials, composition conditions, reaction conditions, and the like. The range can be freely changed within a range in which a contrast agent for acoustic imaging can be obtained.

  In addition, the particle size of the contrast agent for photoacoustic imaging in the following examples is a value measured using a dynamic light scattering analyzer (DLS-8000, manufactured by Otsuka Electronics Co., Ltd.). Accumulation of 100 times was performed 5 times using the above apparatus, and a weight average value was calculated by Marquardt analysis, or a cumulant value was calculated by Cumulant analysis. The average of the five calculated data was used as the particle size of the contrast agent. In addition, the particle size of iron oxide particles in the following examples is the particle size of primary particles of iron oxide measured from an image obtained using a transmission electron microscope (TEM).

Example 1
(Relationship between iron oxide particle size, molar extinction coefficient and photoacoustic signal)
For the contrast agent for photoacoustic imaging having iron oxide particles having different particle diameters, the molar extinction coefficient and the photoacoustic signal per mol of iron atoms and per mol of the number of particles were measured.

  As a contrast agent for photoacoustic imaging having iron oxide particles having different particle diameters, iron oxide particles coated with a polymer and having different primary particle diameters were used. Specifically, the primary particle size is 5 nm (iron atom concentration 1.45 mg / ml, iron oxide particle concentration 10 nmol / ml, SHP-05, manufactured by Ocean NanoTech), 10 nm (iron atom concentration 5 mg / ml, Iron oxide particle concentration 4.3 nmol / ml, SHP-10, manufactured by Ocean NanoTech), 15 nm (iron atom concentration 5 mg / ml, iron oxide particle concentration 1.25 nmol / ml, SHP-15, manufactured by Ocean NanoTech), 20 nm (Iron atom concentration 5 mg / ml, iron oxide particle concentration 0.55 nmol / ml, SHP-20, manufactured by Ocean NanoTech) and 50 nm (iron atom concentration 5 mg / ml, iron oxide particle concentration 0.034 nmol / ml, SHP- 50, manufactured by Ocean NanoTech), and 100 nm (described later) The iron oxide particles which are the particles of Example 10) were used.

  These iron oxide particles are dispersed in water, and the iron oxide particles are formed in a single particle state. Absorbance was measured at 710, 750, 800, and 850 nm using a spectrophotometer (Lambda Bio40, manufactured by Perkin elmer). The measurement container was a polystyrene cuvette with a width of 1 cm and an optical path length of 0.1 cm. In this container, 200 μl of a dispersion of iron oxide particles was put and the absorbance was measured.

  The molar extinction coefficient per mole of iron atom and the molar extinction coefficient per mole of iron oxide particles were calculated from the absorbance, iron atom concentration and particle concentration.

The photoacoustic signal is measured by irradiating a contrast medium for photoacoustic imaging dispersed in water with pulsed laser light, detecting the photoacoustic signal from the contrast medium using a piezoelectric element, amplifying it with a high-speed preamplifier, and then using a digital oscilloscope. The waveform was acquired. Specific conditions are as follows. A titanium sapphire laser (LT-2211-PC, manufactured by Lotis) was used as a pulsed laser light source. The wavelengths were 710, 750, 800, and 850 nm, the energy density was 20-50 mJ / cm ² (depending on the selected wavelength), the pulse width was about 20 nanoseconds, and the pulse repetition frequency was 10 Hz. The polystyrene cuvette was used as a measurement container for containing a contrast agent for photoacoustic imaging dispersed in water. As the piezoelectric element for detecting the photoacoustic signal, a non-convergent ultrasonic transducer (V303, manufactured by Panametrics-NDT) having an element diameter of 1.27 cm and a center band of 1 MHz was used. The measurement container and the piezoelectric element were immersed in a glass container filled with water, and the interval was set to 2.5 cm. As the high-speed preamplifier for amplifying the photoacoustic signal, an ultrasonic preamplifier (Model 5682, manufactured by Olympus) having an amplification degree of +30 dB was used. The amplified signal was input to a digital oscilloscope (DPO4104, manufactured by Tektronix). The polystyrene cuvette was irradiated with pulsed laser light from the outside of the glass container. A part of the scattered light generated at this time was detected by a photodiode and input as a trigger signal to a digital oscilloscope. The digital oscilloscope was set to an average display mode for 32 times, and an average photoacoustic signal was acquired 32 times for laser pulse irradiation. FIG. 4 shows a typical photoacoustic signal waveform. As indicated by the arrows in the figure, the photoacoustic signal intensity (V) was obtained from the waveform. A value obtained by dividing the obtained photoacoustic signal intensity by the energy (J) of the irradiation pulse laser was defined as a normalized photoacoustic signal (VJ ⁻¹ ), and used as evaluation means hereinafter.

  The normalized photoacoustic signal per mole of iron atoms and the normalized photoacoustic signal per mole of iron oxide particles were calculated from the iron atom concentration and the particle concentration, respectively.

  FIG. 5 shows the molar extinction coefficient and photoacoustic signal of iron oxide particles having different particle sizes. 5A shows the molar extinction coefficient per mol of iron atoms contained in the iron oxide particles at 710 nm, and FIG. 5B shows the molar extinction coefficient per mol of iron oxide particles at 710 nm. As the particle size of the iron oxide particles increased, the molar extinction coefficient per mole of iron atoms tended to increase. Even at other wavelengths, an increase in the molar extinction coefficient per mole of iron atoms was observed. Due to this effect, the molar extinction coefficient per 1 mol of iron oxide particles was remarkably increased as the particle size of the iron oxide particles was increased.

  FIG. 5 (c) shows the normalized photoacoustic signal per mole of iron atoms contained in the iron oxide particles at 710 nm, and FIG. 5 (d) shows the normalized photoacoustic signal per mole of iron oxide particles at 710nm. Show. As the particle size of the iron oxide particles increased, the normalized photoacoustic signal per mole of iron atoms increased. Similarly, at other wavelengths, the normalized photoacoustic signal per mole of iron atoms increased. Due to this effect, the normalized photoacoustic signal per 1 mol of iron oxide particles significantly increased as the particle size of the iron oxide particles increased.

  As the particle diameter of the iron oxide particles increased, the amount of light absorbed increased, and the photoacoustic signal emitted from the iron oxide particles increased accordingly. In particular, in iron oxide particles of 15 nm or more, an increase in light absorption and an increase in photoacoustic signal were observed.

(Example 2)
(Preparation of contrast agent 1 for photoacoustic imaging (NP1))
A contrast agent for photoacoustic imaging comprising a polyoxyethylene sorbitan fatty acid ester, a hydrophobic polymer, and iron oxide particles was prepared by the following method.

  50 nm iron oxide particles coated with oleic acid (13.8 mg, SOR-50, manufactured by Ocean NanoTech) and a copolymer of polylactic acid and glycolic acid (9.2 mg, MW 20000, lactic acid: glycolic acid) = 1: 1, PLGA5020, manufactured by Wako Pure Chemical Industries, Ltd.) was added to chloroform (1 ml) and irradiated with an ultrasonic bath for 10 minutes to prepare a chloroform solution.

  Next, the chloroform solution was added to an aqueous solution (12 ml) in which Tween 20 (polyoxyethylene sorbitan monolaurate, 60 mg, manufactured by Kishida Chemical Co., Ltd.) was dissolved to prepare a mixed solution.

  Thereafter, an O / W type emulsion was prepared by treating with an ultrasonic disperser (Ultrasonic disruptor UD-200, manufactured by Tomy) for 4 minutes.

  Next, the emulsion was depressurized at 40 ° C. and 100 hPa for 2 hours, and chloroform was distilled off from the dispersoid to obtain an aqueous dispersion of contrast agent 1 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 1 for photoacoustic imaging may be abbreviated as NP1.

(Example 3)
(Preparation of contrast agent 2 for photoacoustic imaging (NP2))
A contrast agent for photoacoustic imaging composed of phospholipid, polyoxyethylene sorbitan fatty acid ester, hydrophobic polymer, and iron oxide particles was prepared by the following method.

  50 nm iron oxide particles coated with oleic acid (13.8 mg, SOR-50, manufactured by Ocean NanoTech) and a copolymer of polylactic acid and glycolic acid (9.2 mg) were added to chloroform (1 ml), Irradiated with a sonic bath for 10 minutes to prepare a chloroform solution.

  Next, an aqueous solution (12 ml) in which Tween 20 (60 mg) and a phospholipid (7.3 mg, DSPE-PEG-MAL, SUNBRIGHT DSPE-020MA, manufactured by NOF Corporation) having a maleimide group at the end represented by Chemical Formula 4 were dissolved, were dissolved. The chloroform solution was added to obtain a mixed solution.

  Thereafter, an O / W type emulsion was prepared by treating with an ultrasonic disperser for 4 minutes.

  Next, the emulsion was decompressed at 40 ° C. and 100 hPa for 2 hours, and chloroform was distilled off from the dispersoid to obtain an aqueous dispersion of contrast agent 2 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 2 for photoacoustic imaging may be abbreviated as NP2.

(Physical property evaluation of NP1 and NP2)
NP1 and NP2 were dispersed in water, and the particle size was measured using the dynamic light scattering analyzer. The results are shown in FIGS. 6 (a) and 7 (a). The particle sizes (in terms of weight) of NP1 and NP2 were 205 and 176 nm, respectively.

  NP1 and NP2 were observed using a transmission electron microscope (TEM, H800, manufactured by Hitachi). FIG. 6B and FIG. 7B are TEM photographs of the contrast agent for photoacoustic imaging. A contrast agent for photoacoustic imaging having a plurality of iron oxide particles was observed.

  The amount of iron oxide particles contained in NP1 and NP2 was quantified using an emission spectrometer (ICP, CIROS CCD, manufactured by SPECTRO). The contrast agent for photoacoustic imaging was dissolved with concentrated nitric acid, the amount of Fe was quantified with ICP, and the mass of iron oxide particles was calculated. The aqueous dispersion of the contrast agent for photoacoustic imaging was freeze-dried, and then the mass was measured to determine the mass of the entire contrast agent for photoacoustic imaging. When the content of iron oxide particles in the contrast agent for photoacoustic imaging was calculated from the mass of the iron oxide particles and the contrast agent for photoacoustic imaging, NP1 and NP2 were 58 and 63 (wt)%, respectively. It was. A contrast agent for photoacoustic imaging containing iron oxide particles at a high rate was obtained.

The light absorbability of the obtained NP1 and NP2 was evaluated by determining the molar extinction coefficient per mole of particles of the contrast agent for photoacoustic imaging. The light absorption of NP1 and NP2 dispersed in water was measured using a spectrophotometer. The particle concentration of the contrast agent for photoacoustic imaging was calculated from the physical property values obtained by the above method. Table 1 summarizes the molar extinction coefficient per mole of particles of the contrast agent for photoacoustic imaging at wavelengths of 710, 750, 800, and 850 nm. The molar extinction coefficients of NP1, NP2, and Rhizovist (registered trademark) at a wavelength of 710 nm are 2.1 × 10 ¹⁰ , 9.9 × 10 ⁹ , 1.6 × 10 ⁶ (M ⁻¹ cm ⁻¹ (number of particles), respectively. 1 mol)). A contrast agent for photoacoustic imaging having superior light absorption as compared with Rhizovist (registered trademark) was obtained.

Table 2 shows normalized photoacoustic signals per particle of NP1 and NP2. The normalized photoacoustic signals per mole of NP1, NP2, and Rhizovist (registered trademark) particles at a wavelength of 710 nm are 3.5 × 10 ¹¹ , 2.0 × 10 ¹¹ , 6.0 × 10 ⁷ (VJ), respectively. ⁻¹ M ⁻¹ (number of particles 1 mol)). A contrast agent for photoacoustic imaging that emits a strong acoustic signal was obtained as compared with Rhizovist (registered trademark), which is a known contrast agent for photoacoustic imaging.

(Evaluation of stability of NP2)
The stability of NP2 was evaluated by the following method. First, NP2 was stored in water at 4 ° C., and the particle size (cumulant value) and photoacoustic signal of NP2 were measured at regular intervals.

  FIG. 8A shows the result of measuring the particle size of NP2 over time. There was no change in the particle size of NP2, and no aggregation was observed. FIG. 8B shows the results of measuring the photoacoustic signal of NP2 over time when light with a wavelength of 710 nm is irradiated. Even when stored for a long period of time, the normalized photoacoustic signal per mole of iron atom of NP2 was not changed and showed excellent storage stability.

(Quantification of phospholipids on NP2)
The amount of phospholipid introduced to the surface of NP2 was quantified using the activity of the terminal maleimide group. NP2 and L-cysteine were mixed and incubated overnight to allow a covalent reaction between the maleimide group on NP2 and the thiol group on L-cysteine. Unreacted L-cysteine was recovered by precipitating NP2 by centrifugation and recovering the supernatant. Unreacted L-cysteine was quantified by mixing with 5,5′-Dithiobis (2-nitrobenzoic acid) and measuring the absorbance at 420 nm. The amount of phospholipid on one NP2 was 5.6 × 10 ⁵ cells / piece.

Example 4
(Preparation of single chain antibody hu4D5-8scFv)
Based on the gene sequence (hu4D5-8) of the IgG variable region that binds to HER2, a gene hu4D5-8scFv encoding a single chain antibody (scFv) was prepared. First, cDNA was prepared by linking the VL and VH genes of hu4D5-8 with cDNA encoding peptide (GGGGS) ₃ . A restriction enzyme NcoI- was introduced at the 5 'end and a restriction enzyme NotI recognition site was introduced at the 3' end. The base sequence is shown below.
5'-CCATGGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGATCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGCGG TGGTGGCAGCGGTGGCGGTGGCAGCGGCGGTGGCGGTAGCGAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAAGGATTTATCCTACGAATGGTTATACTAGATATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTGCAGATGAACAGCCTGCGTGCTGAGGACACTGCCGTCTATTATTGTTCTAG TGGGGAGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCGGCCGC-3 '(SEQ ID NO: 1)
The sequence CCATG from the 5 ′ end to the 5th amino acid and the sequence GCGGCCGC from the 5 ′ end to the 8th amino acid represent the restriction enzyme recognition site.

  The gene fragment hu4D5-8scFv was inserted downstream of the T7 / lac promoter of the plasmid pET-22b (+) (Novagen). Specifically, the above cDNA is ligated to pET-22b (+) (manufactured by Novagen) digested with restriction enzymes NcoI- and NotI.

This expression plasmid was transformed into Escherichia coli BL21 (DE3) to obtain an expression strain. The obtained strain was precultured overnight in 4 ml of LB-Amp medium, and the whole amount was added to 250 ml of 2 × YT medium, and cultured with shaking at 28 ° C. and 120 rpm for 8 hours. Thereafter, IPTG (Isopropyl-β-D (−)-thiogalactopylanoside) was added at a final concentration of 1 mM, and the cells were cultured at 28 ° C. overnight. The cultured Escherichia coli was centrifuged at 8000 × g for 30 minutes at 4 ° C., and the supernatant culture was collected. Ammonium sulfate of 60% by weight of the obtained culture broth was added, and the protein was precipitated by salting out. The salted-out solution was allowed to stand overnight at 4 ° C., and then centrifuged at 8000 × g for 30 minutes at 4 ° C. to collect a precipitate. The resulting precipitate was dissolved in 20 mM Tris · HCl / 500 mM NaCl buffer and dialyzed against 1 liter of the same buffer. The protein solution after dialysis was added to a column packed with His / Bind (registered trademark) Resin (manufactured by Novagen) and purified by metal chelate affinity chromatography via Ni ions. The purified hu4D5-8scFv showed a single band by reducing SDS-PAGE and confirmed that the molecular weight was about 28 kDa. The amino acid sequence of the prepared antibody is shown below. Hereinafter, hu4D5-8 scFv may be abbreviated as scFv.
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAAALEHHHHHHGGC (SEQ ID NO: 2)
(Binding of single chain antibody hu4D5-8scFv and NP2)
Phosphate buffer containing 5 mM EDTA the scFv prepared above (2.68 mM KCl / 137 mM NaCl / 1.47 mM KH 2 PO 4 / 1m M Na 2 HPO 4/5 mM EDTA, pH7.4) to After buffer replacement, reduction treatment was performed at 25 ° C. for about 2 hours with 20 times the amount of scFv in terms of tri (2-carboxyethyl) phosphine hydrochloride (TCEP). This reduced scFv was reacted with NP2 prepared above at 25 ° C. for 3 hours. The scFv of 2000 times the molar amount of NP2 particles was used in the reaction. Centrifugation gave a precipitate of NP2 bound to scFv and an unreacted scFv supernatant. Unreacted scFv was quantified by liquid chromatography. The scFv bound on one grain of NP2 was 1.4 × 10 ³ / piece.

(Example 5)
(Preparation of contrast agent 3 for photoacoustic imaging (NP3))
A contrast agent for photoacoustic imaging composed of an amphiphilic polymer and iron oxide particles was prepared by the following method.

A styrene-maleic anhydride copolymer (12.5 mg, average molecular weight: 9500, styrene: maleic anhydride = 75: 25 (molar ratio), manufactured by Polysciences) was dissolved in chloroform (3 ml), and then MeO-PEG was dissolved therein. -NH ₂ (45 mg, average molecular weight: 5000, SUNBRIGHT ME-50EA, manufactured by NOF CORPORATION) by reacting the addition of amphiphilic polymer (styrene was coupled PEG - maleic anhydride copolymer) to give the It was.

  50 nm iron oxide particles coated with oleic acid (14 mg, SOR-50, manufactured by Ocean NanoTech) and the above amphiphilic polymer (50 mg) were added to chloroform (3 ml), and then the chloroform was distilled off by evaporation. did.

  Thereafter, water (18 ml) was added and the mixture was treated with an ultrasonic bath for 1 minute to obtain an aqueous dispersion of the contrast agent 3 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 3 for photoacoustic imaging may be abbreviated as NP3.

The particle size of NP3 was 211 nm (weight conversion). Moreover, the molar extinction coefficient per 1 mol number of particles of NP3 is 1.9 × 10 ¹⁰ (M ⁻¹ cm ⁻¹ (1 mol number of particles)), and the normalized photoacoustic signal per 1 mol number of particles is 4 .8 × 10 ¹¹ (V J ⁻¹ M ⁻¹ (number of particles 1 mol)).

  NP3 was observed using cryo-TEM, and the result is shown in FIG. 9 (a). From FIG. 9A, it was confirmed that NP3 contained a plurality of iron oxide particles.

  NP3 was observed using a cryo-FIB / SEM, and the results are shown in FIG. 9 (b). In FIG. 9B, it was confirmed that the iron oxide particles were localized on the particle surface side of NP3 when the acceleration voltage was 2 kV. Further, when the acceleration voltage is increased to 5 kV and 8 kV, iron oxide particles are present on the back side of the annularly arranged iron oxide particles observed at 2 kV (in the reverse direction of the paper surface of this specification), and NP3 has a hollow structure. It was confirmed that

(Example 6)
(Preparation of contrast agent 4 for photoacoustic imaging (NP4))
The contrast agent 4 for photoacoustic imaging composed of an amphiphilic polymer and iron oxide particles was prepared by the following method.

An octadecene-maleic anhydride alternating copolymer (12.5 mg, average molecular weight: 40000, octadecene: maleic anhydride = 50: 50 (molar ratio), manufactured by Aldrich) was dissolved in chloroform (3 ml), and then MeO- PEG-NH ₂ (94 mg, average molecular weight: 10,000, SUNBRIGHT ME-100EA, manufactured by NOF Corporation) was added and reacted to make an amphiphilic polymer (octadecene-maleic anhydride alternating copolymer into which a PEG chain was introduced). Got.

  Iron oxide particles (14 mg, SOR-50, manufactured by Ocean NanoTech) and the above amphiphilic polymer (100 mg) were added to chloroform (3 ml), and then chloroform was distilled off by evaporation.

  Thereafter, water (18 ml) was added and the mixture was treated with an ultrasonic bath for 1 minute to obtain an aqueous dispersion of the contrast agent 4 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 4 for photoacoustic imaging may be abbreviated as NP4.

The particle size of NP4 was 161 nm (weight conversion). Moreover, the molar extinction coefficient per 1 mol number of particles of NP4 is 1.0 × 10 ¹⁰ (M ⁻¹ cm ⁻¹ (1 mol number of particles)), and the normalized photoacoustic signal per 1 mol number of particles is 2. 0.0 × 10 ¹¹ (V J ⁻¹ M ⁻¹ (number of particles 1 mol)).

(Example 7)
(Preparation of photoacoustic contrast agent 5 (NP5))
The contrast agent 5 for photoacoustic imaging comprising an amphiphilic polymer and iron oxide particles was produced in the same manner as in Example 6.

An octadecene-maleic anhydride alternating copolymer (12.5 mg) was dissolved in chloroform (3 ml), and MeO-PEG-NH ₂ (47 mg, average molecular weight: 5000, SUNBRIGHT ME-50EA, manufactured by NOF Corporation) was added thereto. In addition, an amphiphilic polymer (an octadecene-maleic anhydride alternating copolymer into which a PEG chain was introduced) was obtained by reaction.

  Iron oxide particles (14 mg) and the above amphiphilic polymer (50 mg) were added to chloroform (3 ml), and then chloroform was distilled off by evaporation.

  Thereafter, water (18 ml) was added and the mixture was treated with an ultrasonic bath for 1 minute to obtain an aqueous dispersion of the contrast agent 5 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 5 for photoacoustic imaging may be abbreviated as NP5.

The particle size of NP5 was 163 nm (weight conversion). Moreover, the molar extinction coefficient per 1 mol number of particles of NP5 is 1.2 × 10 ¹⁰ (M ⁻¹ cm ⁻¹ (1 mol number of particles)), and the normalized photoacoustic signal per 1 mol number of particles is 2 .8 × 10 ¹¹ (V J ⁻¹ M ⁻¹ (number of particles 1 mol)).

(Example 8)
(Preparation of contrast agent 6 for photoacoustic imaging (NP6))
The contrast agent 6 for photoacoustic imaging composed of phospholipid and iron oxide particles was prepared by the following method.

  Phospholipid (20 mg, SUNBRIGHT DSPE-020CN, manufactured by NOF Corporation) having a methoxy group at the end represented by Chemical Formula 5 was dissolved in chloroform (3 ml).

  Iron oxide particles (14 mg, SOR-50, manufactured by Ocean NanoTech) were added to the above phospholipid chloroform solution, and then chloroform was distilled off by evaporation.

  Thereafter, water (18 ml) was added and the mixture was treated with an ultrasonic bath for 1 minute to obtain an aqueous dispersion of the contrast agent 6 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 6 for photoacoustic imaging may be abbreviated as NP6.

The particle size of NP6 was 183 nm (weight conversion). Moreover, the molar extinction coefficient per 1 mol number of particles of NP6 is 2.5 × 10 ¹⁰ (M ⁻¹ cm ⁻¹ (1 mol number of particles)), and the normalized photoacoustic signal per 1 mol number of particles is 4. 4 × 10 ¹¹ (V J ⁻¹ M ⁻¹ (number of particles 1 mol)).

  NP6 was observed using cryo-TEM, and the results are shown in FIG. From FIG. 10, it was confirmed that NP6 contains a plurality of iron oxide particles.

Example 9
(Preparation of contrast agent 7 for photoacoustic imaging (NP7))
The contrast agent 7 for photoacoustic imaging composed of phospholipid and iron oxide particles was produced in the same manner as in Example 8.

  Phospholipid (20 mg, DSPE-020CN, manufactured by NOF CORPORATION) having a methoxy group at the end represented by Chemical Formula 5 was dissolved in chloroform (3 ml).

  Iron oxide particles coated with oleic acid (15 mg, particle size 100 nm, prototype manufactured by Ocean NanoTech) were added to the above phospholipid chloroform solution, and the chloroform was distilled off by evaporation.

  Thereafter, water (18 ml) was added and the mixture was treated in an ultrasonic bath for 1 minute to obtain an aqueous dispersion of the contrast agent 7 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 7 for photoacoustic imaging may be abbreviated as NP7.

The particle size of NP7 was 117 nm (weight conversion). Moreover, the molar extinction coefficient per 1 mol number of particles of NP7 is 1.5 × 10 ⁹ (M ⁻¹ cm ⁻¹ (1 mol number of particles)), and the normalized photoacoustic signal per 1 mol number of particles is 2 .6 × 10 ¹⁰ (V J ⁻¹ M ⁻¹ (number of particles 1 mol)).

  NP7 was observed using TEM, and the results are shown in FIG. FIG. 11B is an enlarged view of FIG. From FIG. 11, it was confirmed that in NP7, one iron oxide particle was coated with phospholipid.

(Example 10)
(Preparation of contrast agent 8 for photoacoustic imaging (NP8))
The contrast agent 8 for photoacoustic imaging composed of an amphiphilic polymer and iron oxide particles was prepared by the following method.

  To the terminal carboxylic acid of PLGA-PEG (35 mg, PLGA (lactic acid-glycolic acid copolymer, average molecular weight 20000, PLGA-5020, manufactured by Wako Pure Chemical Industries, Ltd.)) in chloroform (3 ml), PEG (average molecular weight 5000, SUNBRIGHT MEPA- 50H, manufactured by NOF Corporation) was dissolved in an amide-bonded amphiphilic polymer, in-house synthesized product).

  Iron oxide particles (16 mg, SOR-50, manufactured by Ocean NanoTech) were added to the above PLGA-PEG chloroform solution, and then uniformly dispersed.

  Thereafter, water (13 ml) was added and the mixture was treated with an ultrasonic disperser for 4 minutes to prepare an O / W type emulsion.

  Next, the emulsion was depressurized at 40 ° C. and 100 hPa for 2 hours, and chloroform was distilled off from the dispersoid to obtain an aqueous dispersion of contrast agent 8 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 8 for photoacoustic imaging may be abbreviated as NP8.

The particle size of NP8 was 213 nm (weight conversion). Moreover, the molar extinction coefficient per 1 mol number of particles of NP8 is 1.3 × 10 ¹⁰ (M ⁻¹ cm ⁻¹ (1 mol number of particles)), and the normalized photoacoustic signal per 1 mol number of particles is 1. .8 × 10 ¹¹ (V J ⁻¹ M ⁻¹ (number of particles 1 mol)).

(Example 11)
(Preparation of contrast agent 9 for photoacoustic imaging (NP9))
A contrast agent 9 for photoacoustic imaging composed of an amphiphilic polymer and iron oxide particles was produced in the same manner as in Example 10.

  PLGA-PEG (31 mg, amphiphilic polymer in which PEG (average molecular weight 2000, SUNBRIGHT MEPA-20H, manufactured by NOF Corporation) is amide-bonded in the terminal carboxylic acid of PLGA-5020) dissolved in chloroform (3 ml) did.

  Iron oxide particles (16 mg, SOR-50, manufactured by Ocean NanoTech) were added to the above PLGA-PEG chloroform solution, and then uniformly dispersed.

  Thereafter, water (13 ml) was added and the mixture was treated with an ultrasonic disperser for 4 minutes to prepare an O / W type emulsion.

  Next, the emulsion was depressurized at 40 ° C. and 100 hPa for 2 hours, and chloroform was distilled off from the dispersoid to obtain an aqueous dispersion of contrast agent 9 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 9 for photoacoustic imaging may be abbreviated as NP9.

The particle size of NP9 was 441 nm (weight conversion). Moreover, the molar extinction coefficient per 1 mol number of particles of NP9 is 8.1 × 10 ¹⁰ (M ⁻¹ cm ⁻¹ (1 mol number of particles)), and the normalized photoacoustic signal per 1 mol number of particles is 8 7 × 10 ¹¹ (V J ⁻¹ M ⁻¹ (number of particles 1 mol)).

(Example 12)
(Preparation of contrast agent 10 for photoacoustic imaging (NP10))
A photoacoustic contrast agent 10 (NP10) composed of an amphiphilic polymer, a hydrophobic polymer, and iron oxide particles was produced by the following method.

  50 nm iron oxide particles coated with oleic acid (13.8 mg, SOR-50, manufactured by Ocean Nanotech) and a copolymer of polylactic acid and glycolic acid (9.2 mg) were added to chloroform (1 ml), Irradiated with an ultrasonic bath for 10 minutes to prepare a chloroform solution.

  Next, the chloroform solution was added to 12 ml of an aqueous solution in which polyvinyl alcohol (480 mg, MW 31000, saponification degree 88%, manufactured by Sigma-Aldrich) was dissolved to obtain a mixed solution.

  Thereafter, an O / W type emulsion was prepared by treating with an ultrasonic disperser for 4 minutes.

  Next, the emulsion was decompressed at 40 ° C. and 100 hPa for 2 hours, and chloroform was distilled off from the dispersoid to obtain an aqueous dispersion of the contrast agent 10 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 10 for photoacoustic imaging may be abbreviated as NP10.

The particle size of NP10 was 96 nm (weight conversion). Moreover, the molar extinction coefficient per 1 mol number of particles of NP10 is 1.3 × 10 ⁹ (M ⁻¹ cm ⁻¹ (1 mol number of particles)), and the normalized photoacoustic signal per 1 mol number of particles is 3 .8 × 10 ¹⁰ (V J ⁻¹ M ⁻¹ (number of particles 1 mol)).

(Example 13)
(Preparation of contrast agent 11 for photoacoustic imaging (NP11))
The photoacoustic contrast agent 11 (NP11) composed of phospholipid, polyoxyethylene sorbitan fatty acid ester, hydrophobic polymer, and iron oxide particles was prepared by the following method.

  50 nm iron oxide particles coated with oleic acid (13.8 mg, SOR-50, manufactured by Ocean Nanotech) and a copolymer of polylactic acid and glycolic acid (9.2 mg) were added to chloroform (1 ml), Irradiated with an ultrasonic bath for 10 minutes to prepare a chloroform solution.

  Next, the chloroform was dissolved in 12 ml of an aqueous solution in which Tween 20 (60 mg) and a phospholipid having a methoxy group represented by Chemical Formula 5 (7.3 mg, DSPE-PEG-CN, SUNBRIGHT DSPE-020CN, manufactured by NOF Corporation) were dissolved. The solution was added to make a mixed solution.

  Thereafter, an O / W type emulsion was prepared by treating with an ultrasonic disperser for 4 minutes.

  Next, the emulsion was depressurized at 40 ° C. and 100 hPa for 2 hours, and chloroform was distilled off from the dispersoid to obtain an aqueous dispersion of the contrast agent 11 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 11 for photoacoustic imaging may be abbreviated as NP11.

The particle size of NP11 was 172 nm (weight conversion). Moreover, the molar extinction coefficient per 1 mol number of particles of NP11 is 9.2 × 10 ⁹ (M ⁻¹ cm ⁻¹ (1 mol number of particles)), and the normalized photoacoustic signal per 1 mol number of particles is 1. .6 × 10 ¹¹ (V J ⁻¹ M ⁻¹ (number of particles 1 mol)).

(Example 14)
(Preparation of contrast agent 12 for photoacoustic imaging (NP12))
A contrast agent 12 for photoacoustic imaging (NP12) composed of two types of phospholipids, polyoxyethylene sorbitan fatty acid ester, hydrophobic polymer, and iron oxide particles was prepared by the following method.

  50 nm iron oxide particles coated with oleic acid (13.8 mg, SOR-50, manufactured by Ocean Nanotech) and a copolymer of polylactic acid and glycolic acid (9.2 mg) were added to chloroform (1 ml), Irradiated with an ultrasonic bath for 10 minutes to prepare a chloroform solution.

Next, Tween 20 (60 mg), a phospholipid having a methoxy group represented by Chemical Formula 5 (DSPE-PEG-CN, SUNBRIGHT DSPE-020CN, manufactured by NOF Corporation), and a terminal having an amino group represented by Chemical Formula 1 lipids _{(DSPE-PEG-NH 2,} SUNBRIGHT DSPE-020PA, manufactured by NOF Corporation) was dissolved in water 12 ml. However, the molar ratio of the phospholipid having a methoxy group at the terminal and the amino group having a terminal amino group is 100: 0, 99.9: 0.1, 99.5: 0.5, 99: 1, 95: 5. 90:10, and the total number of moles was 2.5 × 10 ⁻⁶ mol. The chloroform solution was added to the prepared aqueous solution to obtain a mixed solution.

  Thereafter, an O / W type emulsion was prepared by treating with an ultrasonic disperser for 4 minutes.

  Next, the emulsion was depressurized at 40 ° C. and 100 hPa for 2 hours, and chloroform was distilled off from the dispersoid to obtain an aqueous dispersion of the contrast agent 12 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 12 for photoacoustic imaging may be abbreviated as NP12.

Table 3 shows the particle diameter (cumulant value) and the zeta potential value of the contrast agent 12 for photoacoustic imaging. When the ratio of phospholipids having amino groups at the ends increases when producing a contrast agent for photoacoustic imaging, the zeta potential of the obtained contrast agent for photoacoustic imaging tends to increase, and the surface of the contrast agent for photoacoustic imaging It was shown that more amino groups were introduced. Incidentally, NH2 Table 3 is an amino group NH _2.

(Evaluation of stability of NP12)
Among NP12, the ratio of the phospholipid having a methoxy group at the end and the phospholipid having an amino group at the end is represented by a contrast agent for photoacoustic imaging (hereinafter referred to as NP12 (90:10)) prepared under the condition of CN: NH ₂ = 90: 10. The stability was evaluated by the following method.

  NP12 (90:10) was mixed in water, PBS, and 500 mM borate buffer (Bor, pH 8.5) and stored at 4 ° C. and 37 ° C. The particle size (cumulant value) and absorbance after 1 day and 3 days were measured.

  FIG. 12 shows the results of a stability test of PN12 (90:10) in each buffer. When NP12 (90:10) is stored in PBS, the particle size tends to be slightly larger and may aggregate. However, there was no significant change in particle size in water and Bor, and no aggregation was observed. The absorbance of NP12 (90:10) did not change greatly in any of the buffers, and the effect as a contrast agent for photoacoustic imaging was maintained.

(Example 15)
(Binding of single chain antibody hu4D5scFv and NP12 (90:10))
Hu4D5scFv was bound to NP12 (90:10) by the following method.

After buffer replacement to hu4D5scFv prepared in Example 4 in phosphate buffer _{(2.68mM KCl / 137mM NaCl / 1.47mM} KH 2 PO 4 / 1mM Na 2 HPO 4 / 5mM EDTA, pH7.4) containing 5 mM EDTA, The reduction treatment was carried out with 10-fold molar amount of tri (2-carboxyethyl) phosphine hydrochloride (TCEP) at 25 ° C. for about 2 hours.

Hu4D5scFv was bound via a primary amino group present on the surface of NP12 (90:10). First, 0.03 mg (60 nmol) of succinimidyl-[(N-maleimidepropionamido) -diethyleneglycol] ester (SM (PEG) ₂ , Thermo Scientific) was added to an aqueous dispersion of NP12 (90:10) (particle concentration: 3.6 × 10 ¹² / ml) was dissolved in 1.0 ml. Next, 0.11 ml of 500 mM borate buffer (pH 8.5) was added. After stirring this particle suspension at room temperature for 2 hours, NP12 (90:10) into which maleimide groups were introduced (hereinafter referred to as maleimidated NP12) using a PD-10 desalting column (manufactured by GE Healthcare Bioscience). And unreacted SM (PEG) ₂ was separated using water as a developing solvent to obtain approximately 3 ml of an aqueous solution of maleimidated NP12. To this aqueous solution, 60 μl of 1M 2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) solution was added to obtain a HEPES solution of maleimidated NP12.

  The reduced hu4D5scFv was added to the HEPES solution of maleimidated NP12 and reacted at 4 ° C. for 15 hours or longer. The reaction molar ratio (hu4D5scFv / maleimidated NP12) was 1125. Here, “preparation” means added to the reaction system, and “reaction molar ratio of preparation” refers to the molar concentration ratio of hu4D5scFv and maleimidated NP12 added to the reaction system. After the reaction, 6.8 nmol of polyethylene glycol having a terminal thiol group (molecular weight 1000, PLS-606, manufactured by Creative PEGWorks) was added to this solution, and the mixture was stirred at room temperature for 30 minutes. Next, after filtering this solution (pore size 1.2 μm), hu4D5scFv that did not bind to maleimidated NP12 was removed by ultrafiltration using a 100 kDa pore size Amicon Ultra-4 (Nihon Millipore), NP12 (90:10) modified with hu4D5scFv was obtained.

  As a result of calculating the modification amount of hu4D5scFv to NP12 (90:10) using the BCA method, it was found that 847 scFvs were modified per particle.

(Example 16)
(Cancer accumulation of NP12 (90:10))
Cancer accumulation (EPR effect) in tumor-bearing mice of NP12 (90:10) was confirmed by the following method.

NP12 (90:10) was labeled with a radioisotope (hereinafter abbreviated as RI) according to a known method. 125-iodine (may be abbreviated as ¹²⁵ I) was used as the RI nuclide. ¹²⁵ I particle labeling was performed according to the SIB method. A ¹²⁵ I-labeled NP12 (90:10) solution was obtained by purification with PBS using PD-10 (manufactured by GE Healthcare Japan).

Nude mice transplanted with Colon 26 cancer cells were administered 1.8 × 10 ¹¹ mice (0.45 mg in terms of iron amount) ¹²⁵ I-labeled NP12 (90:10) from the tail vein of nude mice. . Nude mice were sacrificed 1, 3, 24, and 72 hours after administration, and blood and cancer radioactivity was measured. The accumulation rate (% ID / g) was determined from the ratio to the dose and the weight of blood and cancer (FIG. 13). ¹²⁵ I-labeled NP12 (90:10) disappeared from the blood with the lapse of time after administration, but accumulation in cancer was observed 3 hours after administration.

(Example 17)
(Preparation of contrast agent 13 for photoacoustic imaging (NP13))
The photoacoustic contrast agent 13 (NP13) composed of phospholipid, polyoxyethylene sorbitan fatty acid ester, hydrophobic polymer, and iron oxide particles was prepared by the following method.

  50 nm iron oxide particles coated with oleic acid (13.8 mg, SOR-50, manufactured by Ocean Nanotech) and a copolymer of polylactic acid and glycolic acid (9.2 mg) were added to chloroform (1 ml), Irradiated with an ultrasonic bath for 10 minutes to prepare a chloroform solution.

  Next, the chloroform solution was added to 12 ml of an aqueous solution in which Tween 20 (600 mg) and a phospholipid having a methoxy group represented by Chemical Formula 5 (73 mg, DSPE-PEG-CN, SUNBRIGHT DSPE-020CN, manufactured by NOF Corporation) were dissolved. In addition, a mixed solution was obtained.

  Thereafter, an O / W type emulsion was prepared by treating with an ultrasonic disperser for 4 minutes.

  Next, the emulsion was decompressed at 40 ° C. and 100 hPa for 2 hours, and chloroform was distilled off from the dispersoid to obtain an aqueous dispersion of the contrast agent 13 for photoacoustic imaging. Purified by centrifugation. Hereinafter, the obtained contrast agent 13 for photoacoustic imaging may be abbreviated as NP13.

The particle size of NP13 was 70 nm (weight conversion). In addition, the molar extinction coefficient per 1 mol of NP13 is 1.2 × 10 ⁹ (M ⁻¹ cm ⁻¹ (1 mol of particles)), and the normalized photoacoustic signal per 1 mol of particles is 3 0.0 × 10 ¹⁰ (V J ⁻¹ M ⁻¹ (number of particles 1 mol)). Compared to Example 13, in Example 17, the concentrations of Tween 20 and phospholipid were increased 10 times. The particle size of the photoacoustic imaging contrast agent could be controlled by the concentration of Tween 20 and phospholipid.

(Example 18)
(Particle size and photoacoustic signal of contrast agent for photoacoustic imaging)
Contrast agents for photoacoustic imaging having different particle diameters were prepared by the following method. As iron oxide particles, 50 nm iron oxide particles (SOR-50, manufactured by Ocean NanoTech) coated with oleic acid, 20 nm iron oxide particles (SOR-20, manufactured by Ocean NanoTech), and oleic acid were coated. 5 nm iron oxide particles (SOR-05, manufactured by Ocean NanoTech) were used.

  5 nm, 20 nm, and 50 nm iron oxide particles (13.8 mg) and a copolymer of polylactic acid and glycolic acid (9.2 mg) were added to chloroform (1 ml) and irradiated with an ultrasonic bath for 10 minutes. A solution was prepared.

  Next, phospholipid was not added to an aqueous solution (12 ml) in which Tween 20 (60 mg, manufactured by Kishida Chemical Co.) was dissolved. When phospholipid was added, the type of functional group at the end of the phospholipid was changed, and various types Of Tween 20 was prepared, and the chloroform solution was added to prepare a mixed solution. Thereafter, the same method as that for NP1 and NP2 was used.

  From the TEM observation and the content of iron oxide particles in the contrast agent for photoacoustic imaging, it was confirmed that the obtained contrast agent for photoacoustic imaging was densely packed with a plurality of iron oxide particles. FIG. 14 shows the relationship between the photoacoustic signal and the particle size (weight conversion) of the contrast agent for photoacoustic imaging containing 50 nm, 20 nm, and 5 nm iron oxide particles. As a photoacoustic signal, FIG. 14A shows a normalized photoacoustic signal per mole of iron atoms, and FIG. 14B shows a normalized photoacoustic signal per mole of contrast agent particles for photoacoustic imaging. Yes. In the contrast agent for photoacoustic imaging containing 50 nm and 20 nm iron oxide particles, an increase in the photoacoustic signal per mole of iron atoms was observed when the particle size of the contrast agent for photoacoustic imaging was increased. The tendency was not related to the production method, that is, the components other than the iron oxide particles. In addition, due to this tendency, in the contrast agent for photoacoustic imaging containing 50 nm iron oxide particles, the contrast agent for photoacoustic imaging per 1 mol of the contrast agent for photoacoustic imaging increases, and a strong photoacoustic signal is generated. An emitted contrast agent for photoacoustic imaging was obtained.

1 Contrast agent for photoacoustic imaging 2 Iron oxide particles 3 Amphiphilic compounds

Claims (10)

  The contrast agent for photoacoustic imaging which has an iron oxide particle, The particle size of the said iron oxide particle is 15 nm or more and 500 nm or less, The contrast agent for photoacoustic imaging characterized by the above-mentioned.   The contrast agent for photoacoustic imaging which has an iron oxide particle, The particle size of the said iron oxide particle is 20 nm or more and 500 nm or less, The contrast agent for photoacoustic imaging characterized by the above-mentioned.   The iron oxide particles are coated with at least one of a homopolymer composed of a monomer having a hydroxycarboxylic acid having 6 or less carbon atoms, or a copolymer composed of two or more kinds of the monomers. The contrast agent for photoacoustic imaging according to claim 1 or 2.   The contrast agent for photoacoustic imaging according to any one of claims 1 to 3, further comprising an amphiphilic compound, wherein the amphiphilic compound is composed of phospholipid, polyoxyethylene sorbitan fatty acid ester, and amphiphilic polymer. The contrast agent for photoacoustic imaging according to any one of claims 1 to 3, wherein the contrast agent is at least one selected amphiphilic compound.   The contrast agent for photoacoustic imaging according to claim 4, wherein the polyoxyethylene sorbitan fatty acid ester is at least one polyoxyethylene sorbitan fatty acid ester selected from Tween 20, Tween 40, Tween 60, and Tween 80. .   The contrast agent for photoacoustic imaging according to claim 4, wherein the phospholipid has a phosphatidyl-based phospholipid having PEG.   Octadecene-maleic anhydride alternating copolymer to which the amphiphilic polymer is bound PEG, styrene-maleic anhydride copolymer to which PEG is bound, polylactic acid to which PEG is bound, lactic acid to which PEG is bound 5. The contrast agent for photoacoustic imaging according to claim 4, wherein the contrast agent is at least one polyoxyethylene sorbitan fatty acid ester selected from a glycolic acid copolymer, polyoxyethylene polyoxypropylene, and polyvinyl alcohol.   The contrast agent for photoacoustic imaging according to any one of claims 5 to 7, wherein a capture molecule is bound to at least a part of the amphiphilic compound.   The contrast agent for photoacoustic imaging according to claim 8, wherein the capture molecule is a single chain antibody. Irradiating the specimen to which the contrast agent for photoacoustic imaging according to claim 1 is administered with light having a wavelength of at least one of 600 nm to 1300 nm;
Detecting an acoustic wave generated from the contrast agent present in the specimen;
A photoacoustic imaging method comprising: