JP6711601B2 - Contrast agent for photoacoustic imaging - Google Patents

Description

The present invention relates to a contrast agent for photoacoustic imaging.

An optical imaging method is known as a method for noninvasively visualizing information inside a living body. In the optical imaging method, signals such as acoustic waves and fluorescence emitted by a substance (light absorber) that absorbs light inside a measured object when the measured object is irradiated with light are measured and imaged. A technique of irradiating a subject with light and measuring an acoustic wave to form an image is called photoacoustic imaging (hereinafter, may be abbreviated as PAI).

In addition, an organic dye that absorbs light in the near-infrared wavelength region, such as indocyanine green (hereinafter sometimes abbreviated as ICG), may be administered to a living body to measure acoustic waves emitted by the organic dye in the living body. It is possible. Therefore, an organic dye such as ICG can be used as a contrast agent for photoacoustic imaging.

Here, in order to use the organic dye as a tumor contrast agent, it is preferable to bind a high molecular compound such as a polymer or protein to the organic dye. Because organic dyes are generally small molecules, they are rapidly excreted from the blood before they accumulate in tumors, but by binding a high molecular compound it becomes difficult to excrete them and they circulate in the blood. This is because the tumor accumulates in the tumor over time, and as a result, the tumor accumulation amount can be increased.

In Patent Document 1, a derivative of ICG is bound to human serum albumin (human serum albumin, hereinafter sometimes abbreviated as HSA) to form a complex, which makes it difficult to be excreted from the blood.

JP, 2014-129332, A

Here, in photoacoustic imaging, it is preferable to obtain a larger photoacoustic signal in order to obtain an image in which the tumor site becomes clearer. A large signal can be obtained by increasing the amount of the organic dye contained in the contrast agent, but from the viewpoint of the burden on the human body, it is preferable that the amount of the organic dye contained in the contrast agent is small. Therefore, it is an object of the present invention to provide a contrast agent for photoacoustic imaging in which a signal per concentration of an organic dye is larger than in the past.

上記式（１）において、Ｒ_１乃至Ｒ_８は各々独立に同一でも異なっていてもよく、水素原子、炭素数１以上３以下のアルキル基、スルホン酸基のいずれかを表し、Ｒ_９乃至Ｒ_１２は、各々独立に同一でも異なっていてもよく、水素原子、炭素数１以上３以下のアルキル基のいずれかを表し、Ｒ_１３及びＲ_１４は同一でも異なっていてもよく、置換基を有していてもよい炭素数１以上５以下のアルキル基を表し、該置換基はカルボキシル基、スルホン酸基及びホスホン酸基のいずれかであり、ｎ_１及びｎ_２は同一でも異なってもよく、０又は１を表し、Ｌ_１乃至Ｌ_７はメチン基であり、Ｌ_１乃至Ｌ_７は４員環乃至６員環を形成していてもよく、Ｒ_１００は、フェニル基、ピリジル基及び置換基を有していてもよい炭素数１以上５以下のアルキル基の少なくともいずれかを表し、該置換基はアリール基、アリールアルキル基、スルホアルキル基、アルキルチオ基、スルホアルキルチオ基、複合環基、アシルアミノ基、アリールアミノ基、Ｎ−アリール−Ｎ−アルキルアミノ基、アリールチオ基、アリールオキシ基、ハロゲン原子、及びアシルアミノアリールオキシ基のいずれかである。 The contrast agent for photoacoustic imaging according to the present invention is a contrast agent for photoacoustic imaging having a plurality of complexes in which at least one organic dye is covalently bonded to albumin, wherein the organic dye is represented by the following formula (1). Ri compound or a salt thereof der represented dye labeling index in the photoacoustic imaging contrast agent is 4 to 10.

In the above formula (1), R _{1 to} R ₈ may independently be the same or different and each represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or a sulfonic acid group, and R _{9 to} R 8 ₁₂ may independently be the same or different and each represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ₁₃ and R ₁₄ may be the same or different and each have a substituent. Represents an optionally substituted alkyl group having 1 to 5 carbon atoms, the substituent is any one of a carboxyl group, a sulfonic acid group and a phosphonic acid group, n ₁ and n ₂ may be the same or different, 0 or 1, L _{1 to} L ₇ are methine groups, L _{1 to} L ₇ may form a 4-membered ring to a 6-membered ring, and R ₁₀₀ is a phenyl group, a pyridyl group or a substituent. Represents at least one of an alkyl group having 1 to 5 carbon atoms which may have, and the substituent is an aryl group, an arylalkyl group, a sulfoalkyl group, an alkylthio group, a sulfoalkylthio group, a complex ring group, an acylamino group. Group, an arylamino group, an N-aryl-N-alkylamino group, an arylthio group, an aryloxy group, a halogen atom, and an acylaminoaryloxy group.

（１）
上記式（１）において、Ｒ_１乃至Ｒ_８は各々独立に同一でも異なっていてもよく、水素原子、炭素数１以上３以下のアルキル基、スルホン酸基のいずれかを表し、Ｒ_９乃至Ｒ_１２は、各々独立に同一でも異なっていてもよく、水素原子、炭素数１以上３以下のアルキル基のいずれかを表し、Ｒ_１３及びＲ_１４は同一でも異なっていてもよく、置換基を有していてもよい炭素数１以上５以下のアルキル基を表し、該置換基はカルボキシル基、スルホン酸基及びホスホン酸基のいずれかであり、ｎ_１及びｎ_２は同一でも異なってもよく、０又は１を表し、Ｌ_１乃至Ｌ_７はメチン基であり、Ｌ_１乃至Ｌ_７は４員環乃至６員環を形成していてもよく、Ｒ_１００は、フェニル基、ピリジル基及び置換基を有していてもよい炭素数１以上５以下のアルキル基の少なくともいずれかを表し、該置換基はアリール基、アリールアルキル基、スルホアルキル基、アルキルチオ基、スルホアルキルチオ基、複合環基、アシルアミノ基、アリールアミノ基、Ｎ−アリール−Ｎ−アルキルアミノ基、アリールチオ基、アリールオキシ基、ハロゲン原子、及びアシルアミノアリールオキシ基のいずれかである。

(1)
In the above formula (1), R _{1 to} R ₈ may independently be the same or different and each represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or a sulfonic acid group, and R _{9 to} R 8 ₁₂ may independently be the same or different and each represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ₁₃ and R ₁₄ may be the same or different and each have a substituent. Represents an optionally substituted alkyl group having 1 to 5 carbon atoms, the substituent is any one of a carboxyl group, a sulfonic acid group and a phosphonic acid group, n ₁ and n ₂ may be the same or different, 0 or 1, L _{1 to} L ₇ are methine groups, L _{1 to} L ₇ may form a 4-membered ring to a 6-membered ring, and R ₁₀₀ is a phenyl group, a pyridyl group or a substituent. Represents at least one of an alkyl group having 1 to 5 carbon atoms which may have, and the substituent is an aryl group, an arylalkyl group, a sulfoalkyl group, an alkylthio group, a sulfoalkylthio group, a complex ring group, an acylamino group. Group, an arylamino group, an N-aryl-N-alkylamino group, an arylthio group, an aryloxy group, a halogen atom, and an acylaminoaryloxy group.

According to the contrast agent for photoacoustic imaging of the present invention, the photoacoustic signal per concentration of the organic dye can be increased.

It is a result showing the relationship between the charging ratio of the dye (152C) and HSA and the dye labeling rate (D/P) in the examples of the present invention. It is a result showing the relationship between the dye labeling rate (D/P) of the contrast agent for photoacoustic imaging and the photoacoustic signal intensity per concentration of the organic dye in the example of the present invention. It is a result showing the relationship between the dye labeling rate (D/P) of the contrast agent for photoacoustic imaging and the photoacoustic signal intensity per HSA concentration in the example of the present invention. It is a result showing the relationship between the dye labeling rate (D/P) of the contrast agent for photoacoustic imaging and the fluorescence intensity in the example of the present invention.

The contrast agent for photoacoustic imaging (hereinafter sometimes abbreviated as PAI) according to the embodiment of the present invention will be described, but the present invention is not limited thereto.

(Contrast agent for photoacoustic imaging)
The contrast agent for photoacoustic imaging according to the present embodiment has a plurality of complexes in which albumin is covalently bonded to at least one specific organic dye. Since the complex has a specific organic dye, that is, a bulky functional group and an organic dye having high hydrophobicity, it is easy for the complexes to gather with each other, resulting in concentration quenching and obtaining a large photoacoustic signal. It is thought to be possible. Therefore, it is suitable for photoacoustic imaging.

The contrast agent for photoacoustic imaging according to the present embodiment may include a dispersion medium, a pharmacologically acceptable additive (for example, a vasodilator), albumin, and the like in addition to the complex. Further, a capture molecule described below may be further bound to albumin in the present embodiment.

The materials constituting the contrast agent for photoacoustic imaging according to this embodiment will be described in detail below.

(Organic dye)
The organic dye in this embodiment is a compound represented by the following formula (1) or a salt thereof. In the present specification, the salt is preferably a pharmaceutically acceptable salt.

（１）
上記式（１）において、Ｒ_１乃至Ｒ_８は各々独立に同一でも異なっていてもよく、水素原子、炭素数１以上３以下のアルキル基、スルホン酸基のいずれかを表す。好ましくは、Ｒ_１乃至Ｒ_８が水素原子の場合である。

(1)
In the above formula (1), R _{1 to} R ₈ may independently be the same or different and each represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or a sulfonic acid group. Preferably, R _{1 to} R ₈ are hydrogen atoms.

In the above formula (1), R _{9 to} R ₁₂ may be the same or different and each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. Preferably, R _{9 to} R ₁₂ are methyl groups.

In the above formula (1), R ₁₃ and R ₁₄ may be the same or different and represent an alkyl group having 1 to 5 carbon atoms which may have a substituent. This substituent is either a carboxyl group, a sulfonic acid group or a phosphonic acid group. Preferred is a propyl group having a substituent, and a preferred substituent is a carboxyl group.

In the above formula (1), n ₁ and n ₂ may be the same or different and represent 0 or 1, but n ₁ and n ₂ are preferably 1.

In the above formula (1), L _{1 to} L ₇ may be methine groups, and L _{1 to} L ₇ may form a 4-membered ring or a 6-membered ring.

In the above formula (1), R ₁₀₀ represents at least one of a phenyl group, a pyridyl group, and an alkyl group having 1 to 5 carbon atoms which may have a substituent. This substituent is an aryl group, an arylalkyl group, a sulfoalkyl group, an alkylthio group, a sulfoalkylthio group, a complex ring group, an acylamino group, an arylamino group, an N-aryl-N-alkylamino group, an arylthio group, an aryloxy group, It is either a halogen atom or an acylaminoaryloxy group. Preferably, R ₁₀₀ is any of a methyl group, an ethyl group, a phenyl group, a pyridyl group and a benzyl group.

Since the structure represented by the above formula (1) has a bulky substituent in the methine chain, it has a high affinity for albumin. Therefore, when this organic dye is bound to albumin, the organic dye easily approaches albumin, and as a result, many organic dyes can be bound to albumin. When a plurality of R _{1 to} R ₈ are sulfonic acid groups, dispersibility in an aqueous solution is high. Thereby, the reactivity with albumin is improved, and it becomes possible to covalently bond a large number of organic dyes to one albumin molecule. It is considered that, by covalently bonding a large number of organic dyes to one molecule of albumin, the organic dyes or the composites are easily aggregated to cause concentration quenching. For this reason, fluorescence is less likely to be emitted from the organic dye that has absorbed the light, and the energy of the absorbed light is likely to be converted into heat energy, so that the acoustic wave becomes large. In addition, since the structure represented by the above formula (1) has a high affinity for albumin, it is considered that the complexes are covalently bound to albumin, and the complexes are likely to be aggregated via the dye. For this reason, fluorescence is less likely to be emitted from the organic dye that has absorbed the light, and the energy of the absorbed light is likely to be converted into heat energy, so that the acoustic wave becomes large.

The organic dye in this embodiment is represented by, for example, the following formulas (2) to (6).

In the above formulas (2) to (6), X is any one of a hydrogen atom, a sodium atom, a potassium atom and an iodine atom.

In addition, the organic dye in the present embodiment is preferably a compound represented by the following formula (7) (hereinafter sometimes abbreviated as 152C dye) or a salt thereof.

（７）
本実施形態における有機色素は、近赤外波長領域の光を吸収して音響波を発する有機色素であることが好ましい。なお、本明細書において近赤外波長領域の光とは６００ｎｍ乃至１３００ｎｍの波長の光を意味する。

(7)
The organic dye in the present embodiment is preferably an organic dye that absorbs light in the near infrared wavelength region and emits an acoustic wave. In this specification, light in the near infrared wavelength region means light having a wavelength of 600 nm to 1300 nm.

(albumin)
Albumin in the present embodiment is a protein that is present in blood in large amounts (about 35 to 50 g/L), has a molecular size of about 66.5 kDa, and has a total sequence of about 585 amino acids. Albumin plays many roles in the body such as osmotic pressure control. Examples of albumin in the present embodiment include human serum albumin (HSA), bovine serum albumin (BSA), and the like, but modified forms such as HSA or BSA may also be used. Moreover, these fragments may be sufficient. The albumin in the present embodiment is any one of human serum albumin, a human serum albumin variant, a human serum albumin fragment, and a human serum albumin variant fragment, which are considered to have high affinity with the human body. Is preferred. In addition, the albumin in the present embodiment may be an extract from human blood or a product from Escherichia coli or the like. Albumin in the present embodiment has at least 95% or more homology with the entire HSA sequence or a partial sequence extracted from the entire sequence. Albumin has a plurality of lysine residues or free cysteine residues at positions accessible by organic dyes. An example of a chemical bond between albumin and an organic dye is an amide bond between an amino group of a lysine residue of albumin and a carboxyl group of a near infrared dye.

(Dye labeling rate (D/P))
The dye labeling rate in the present embodiment is the average number of organic dyes bound to the complex contained in the photoacoustic imaging contrast agent. In the present embodiment, the dye labeling rate can be calculated from the respective concentrations (organic dye concentration/albumin concentration) of the organic dye and albumin contained in the contrast agent for photoacoustic imaging. The concentration of the organic dye can be calculated from the absorbance at the specific absorption wavelength of the organic dye. As the specific absorption wavelength, for example, 765 nm can be adopted when the compound represented by the above formula (7) is used. Further, the concentration of albumin can be determined by the bicinchonic acid (BCA) assay.

Hereinafter, the dye labeling rate may be abbreviated as D/P (Dye/Protein).
In the contrast agent for photoacoustic imaging according to the present embodiment, D/P is preferably 3 or more and 15 or less, and more preferably 4 or more and 10 or less.

(Complex)
The complex in the present embodiment is formed by covalently bonding at least one organic dye to albumin. Here, the term "covalently binding albumin to an organic dye" means that a site excluding a part of albumin and a site excluding a part of the organic dye are covalently bonded. For example, -NH except for H of the amino group of albumin is covalently bonded to -CO except for OH when the organic dye is represented by the above formula (1) and R ₁₃ or R ₁₄ is a carboxyl group. It means doing.
In addition, the “complex” can be referred to as a “compound” or a “molecule” from another viewpoint. A complex (also referred to as a compound) in which one organic dye represented by the above formulas (2) to (6) is bonded to one albumin can be represented as follows.

In the above formulas (i) to (v), X is any one of a hydrogen atom, a sodium atom, a potassium atom and an iodine atom.

In the above formulas (i) to (v), A represents a structure of albumin excluding one amino group.

(Dispersion medium)
Examples of the dispersion medium in the present embodiment include physiological saline, distilled water for injection, phosphate buffered saline (PBS), and glucose aqueous solution. Further, the contrast agent for optical imaging according to the present embodiment, the contrast agent for photoacoustic imaging according to the present embodiment may be pre-dispersed in the above dispersion medium, or may be made into a kit and administered in vivo. Before doing so, it may be used by dispersing it in a dispersion medium.

(Capture molecule)
The capture molecule in the present embodiment is a substance that specifically binds to a target site such as a tumor or a substance that specifically binds to a substance existing around the target site, such as a biomolecule or a chemical substance such as a drug. Can be arbitrarily selected from. Specific examples include proteins, antibodies, antibody fragments, enzymes, bioactive peptides, glycopeptides, peptides, sugar chains, lipids, and molecular recognition compounds. These substances can be used alone or in combination of two or more. By using a complex in which a capture molecule is chemically bound, it is possible to perform specific detection of a target site, tracking of target substance kinetics, localization, drug efficacy, metabolism and the like.

(Method for preparing complex)
In the present embodiment, albumin and the organic dye can be covalently bonded via a known coupling reaction via an amino group, a thiol group, a carboxyl group, or a hydroxyl group. In particular, it is preferable to bond via an amino group. A plurality of amino groups are present in albumin and react efficiently and selectively in a weak alkaline pH range. The organic dye bound to albumin by the above reaction can be washed and purified by a known protein purification method such as ultrafiltration or size exclusion column chromatography. The bond between albumin and the organic dye may be directly bonded through the amino group, thiol group, carboxyl group, or hydroxyl group present on the surface of albumin and the derivative of the organic dye, or various crosslinking materials (crosslinker) You may connect through.

When reacting the amino group of albumin with the carboxyl group of the organic dye, it is preferable to activate the carboxyl group with N-hydroxysuccinimide before the amide bond. Hereinafter, N-hydroxysuccinimide may be abbreviated as NHS.

Further, the amount of the organic dye charged with respect to the amount of HSA charged is preferably 30 or more and 100 or less, and more preferably 40 or more and 80 or less. This is because the dye labeling rate for albumin can be increased, as will be seen from the examples described later.

(Optical imaging method)
The optical imaging method according to the present embodiment includes fluorescence imaging in which a subject is irradiated with light to measure fluorescence and photoacoustic imaging in which a specimen is irradiated with light to measure an acoustic wave (also referred to as an ultrasonic wave). is there. The contrast agent in each method can be called a fluorescence imaging contrast agent and a photoacoustic imaging contrast agent, respectively.

(Photoacoustic imaging method)
A method of detecting a photoacoustic imaging (PAI) contrast agent according to the present embodiment, which is administered in a living body, using a photoacoustic imaging apparatus will be described. The method for detecting a contrast agent for PAI according to this embodiment has the following steps (a) and (b). However, the photoacoustic imaging method according to the present embodiment may include steps other than the steps described below.

(A) A step of irradiating a specimen to which the contrast agent for PAI according to this embodiment is administered with light in a wavelength region of 600 nm to 1300 nm (b) Detecting an acoustic wave generated from the contrast agent existing in the specimen Step The contrast agent for PAI according to the present embodiment has a step of reconstructing a spatial photoacoustic signal intensity distribution from the wavelength, phase, time information and the like of the acoustic wave obtained in (b) above. May be. Note that three-dimensional image reconstruction can be performed based on the wavelength, phase, and time information of the photoacoustic signal obtained in the step (b). The data obtained by image reconstruction may take any form as long as the positional information of the intensity distribution of the photoacoustic signal can be grasped. For example, the photoacoustic signal strength may be expressed in a three-dimensional space, or the photoacoustic signal strength may be expressed in a two-dimensional plane. It is also possible to acquire information for the same observation target by different imaging methods and acquire the positional correspondence between the information and the photoacoustic intensity distribution.

In the step (a) above, a specimen to which the contrast agent for PAI according to this embodiment is administered by a method such as oral administration or injection can be used.

Further, in the step (b), the device for generating the light for irradiating the sample and the device for detecting the photoacoustic signal emitted from the contrast agent for PAI according to the present embodiment are not particularly limited.

The light source for irradiating the sample with light in the step (b) is not limited as long as it can irradiate the sample with laser pulse light of at least one wavelength selected from the range of 600 nm to 1300 nm. .. Examples of the device for irradiating the laser pulse light include a titanium sapphire laser (LT-2211-PC, manufactured by Lotis), an OPO laser (LT-2214 OPO, manufactured by Lotis), and an alexandrite laser.

The device for detecting the acoustic wave is not particularly limited, and various types can be used. For example, a commercially available photoacoustic imaging device (Nexus128, manufactured by Endra Inc.) can be used.

The imaging method using the contrast agent for PAI according to the present embodiment can image a target site such as a tumor, a lymph node, or a blood vessel through the steps (a) and (b).
Note that PAI is a concept including photoacoustic tomography.

The contrast agent for PAI according to the present embodiment can be accumulated more in the tumor site than in the normal site in the body when administered in the body by utilizing the enhanced permeability and retention (EPR) effect. .. As a result, after the contrast agent is administered into the living body, when the living body is irradiated with light and the acoustic wave from the living body is detected, the acoustic wave emitted from the tumor site is larger than the acoustic wave emitted from the normal site. can do. Therefore, the contrast agent for PAI according to the present embodiment is preferably used for tumor imaging.

Further, the PAI contrast agent according to the present embodiment can also be used for contrast enhancement of lymph nodes. Furthermore, it is particularly preferable to use it as a contrast agent for sentinel lymph nodes. When an organic dye such as ICG is used as a contrast agent for a sentinel lymph node, the organic dye administered to the body is rapidly transferred into the blood and discharged outside the body, which limits the observation time. was there. The contrast agent for PAI according to the present embodiment has a larger molecular size than the organic dye, and is expected to have a longer residence time in the sentinel lymph node as a result of a lower diffusion speed in the tissue. Therefore, the PAI contrast agent according to the present embodiment is preferably used for imaging a lymph node, particularly for imaging a sentinel lymph node.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples, and dyes having similar functions and effects such as materials, composition conditions and reaction conditions. It can be freely changed within the range in which the modified albumin is obtained.

(Calculation of dye labeling rate D/P)
In the examples of the present invention, the dye labeling rate for albumin was calculated by measuring the absorbance of the sample (contrast agent for photoacoustic imaging). The dye concentration in the sample was calculated from the absorbance at the absorption wavelength (765 nm) specific to the dye used. Specifically, the sample was diluted with 0.9% sodium dodecyl sulfate (SDS), the absorbance was measured, and it was calculated from the calibration curve of the dye in SDS prepared in advance. The albumin concentration was calculated from the BCA assay.

(Example)
(Preparation of 152C-HSA (complex))
Human serum albumin (Albumin, from human serum: HSA, manufactured by SIGMA, hereinafter simply referred to as HSA) was dissolved in a carbonate buffer (pH 9.6) at a concentration of 10 mg/mL to prepare an HSA solution.

On the other hand, the compound (152C dye) represented by the above formula (7), N-(3-Dimethylaminopropoxyl)-N'-ethylcarbodiimide hydrochloride (sigma-aldrich) in a 3-fold molar amount of the 152C dye, and 1.52C dye. A 5-fold molar amount of NHS (manufactured by Kishida Chemical Co., Ltd.) was mixed in dimethyl sulfoxide (Dimethyl sulfoxide, hereinafter sometimes abbreviated as DMSO) and left at room temperature for 2 hours to obtain a solution containing 152C dye. Next, the solution containing this 152C dye was added to the HSA solution so that the 152C dye was 2, 5, 10, 20, 40, 60, 80 times the molar amount of HSA, and the mixture was reacted at room temperature for 3 hours. It was The N-(3-Dimethylaminopropoxy)-N'-ethylcarbohydrate hydrochloride is sometimes referred to as a water soluble carbimide (WSC).
The obtained reaction mixture solution was subjected to gel filtration (PD-10, manufactured by GE Healthcare Bioscience) to replace with phosphate buffered saline (PBS), and unreacted substances were separated. As a result, a contrast agent for photoacoustic imaging having a complex (152C-HSA) formed by covalently bonding the 152C dye and HSA was prepared.

FIG. 1 shows the dye labeling rate (D/P) of the contrast agent for photoacoustic imaging obtained when the charged amount (mol) of 152C dye was changed with respect to the charged amount (mol) of HSA.

It can be seen from FIG. 1 that D/P can be increased by increasing the amount of the organic dye (152C dye) charged to HSA. Further, it can be seen that a contrast agent for photoacoustic imaging having a very large D/P can be obtained by setting the amount of the organic dye (152C dye) charged to the amount of HSA charged to 40 or more and 80 or less.

(Comparative example)
(Preparation of ICG-HSA (complex))
As a comparative example, a complex in which an ICG derivative and HSA were covalently bonded was prepared. In order to adjust, first, 1 mg of ICG derivative (ICG-Sulfo-OSu, manufactured by DOJINDO Laboratories) was dissolved in 0.1 mL of DMSO, and a 7-fold molar amount of HSA was added to the above HSA solution, and 37°C was added. And allowed to stand for 1.5 hours. This reaction mixture solution was subjected to gel filtration (PD-10) for substitution with phosphate buffered saline (PBS) and separation of unreacted substances to obtain a compound (ICG-HSA) in which an ICG derivative and HSA were covalently bound. Prepared.

(Measurement of photoacoustic signal strength)
The photoacoustic wave intensity of the contrast agent for photoacoustic imaging containing 152C-HSA and ICG-HSA was measured. The photoacoustic signal intensity is measured by irradiating a sample (contrast agent for photoacoustic imaging) dispersed in PBS with pulsed laser light, detecting the intensity of the photoacoustic signal generated from the sample using a piezoelectric element, and using a high speed preamplifier. After amplification with, it was acquired with a digital oscilloscope. The specific conditions are as follows. A titanium sapphire laser (LT-2211-PC, manufactured by Lotus) was used as a light source. The wavelength is variable from 700 to 1000 nm, and the wavelength near the absorption maximum of the sample was selected at the time of measurement. The energy density was about 10 to 20 mJ/cm ² , the pulse width was about 20 nanoseconds, and the pulse repetition frequency was 10 Hz. The piezoelectric element for detecting the photoacoustic signal is a non-focusing ultrasonic transducer (V303, manufactured by Panametrics-NDT) having an element diameter of 1.27 cm and a central band of 1 MHz. A polystyrene cuvette was used as the measurement container, and the optical path length was 0.1 cm and the sample volume was about 200 μl. The measuring instrument uses DPO4104 (manufactured by Tektronix), and a part of scattered light generated when the sample is irradiated with the trigger: pulsed laser light is detected by the photodiode, and the data acquisition: 128 times (128 pulses) under the average condition. The measurement was performed. The obtained photoacoustic signal intensity was standardized by the laser intensity and the measured dye concentration or HSA concentration in the dye-labeled HSA solution.

FIG. 2 shows the results showing the relationship between the dye labeling rate (D/P) of the contrast agent for photoacoustic imaging containing 152C-HSA and the photoacoustic signal intensity per dye concentration. From FIG. 2, it can be seen that a photoacoustic signal intensity higher than that of ICG-HSA can be obtained in any D/P. Further, from FIG. 2, it was confirmed that 152C-HSA has a photoacoustic signal intensity per dye concentration of about 1.5 times higher than that of 152C dye at high D/P (about 8). This can be explained from FIG. 4, which will be described later, because the fluorescence intensity decreases as the D/P increases. That is, it is considered that the higher the D/P is, the easier the complexes are gathered together, the greater the concentration quenching becomes, and the fluorescence emitted from 152C is quenched, so that the photoacoustic signal intensity is improved.
From FIG. 3, it was found that 152-HSA has a photoacoustic signal intensity per concentration of HSA that is about 3.3 times higher than that of ICG-HSA.

(Measurement of fluorescence intensity)
The fluorescence intensity of 152C-HSA was measured using ODYSSEY (registered trademark) CLx Infrared Imaging System (manufactured by LI-COR). It was prepared so that the concentration of 152C-HSA was 50 nM, and the fluorescence intensity was measured. FIG. 4 shows the relationship between the dye labeling rate (D/P) of 152C-HSA and the fluorescence intensity.

(Evaluation of unreacted dye)
The unreacted dye was evaluated as follows. The prepared 152C-HSA was diluted with an SDS sample buffer, applied to a Tris-Glycine gel, subjected to SDS-PAGE (polyacrylamide gel electrophoresis) electrophoresis, and then subjected to ODYSSEY (registered trademark) CLx Infrared ImagingLysCOR System (Synthesis). The product was used to evaluate the fluorescence intensity of the band of the unreacted dye to evaluate the mixing ratio of the unreacted dye. As a result, it was confirmed that 152C-HSA had a very low contamination ratio of unreacted dye of less than 1%. That is, it can be said that the photoacoustic intensity and the fluorescence intensity from the contrast agent for photoacoustic imaging measured in the above Examples are almost derived from the organic dye covalently bonded to albumin.
(Examination of conditions for preparation of 152C-HSA (complex))
The 152C dye, 1 or 3 times the molar amount of the WSC (manufactured by sigma-aldrich) and 1.5 times the molar amount of the dye, NHS (manufactured by Kishida Chemical Co., Ltd.) are mixed in DMSO, and the mixture is mixed at room temperature for 2 hours When placed, a solution containing the 152C dye was obtained. Next, a solution containing this 152C dye was added to the HSA solution such that the 152C dye was 2, 5, 10 or 20 times the molar amount of HSA, and the mixture was reacted at room temperature for 3 hours. As a result, under the condition that the WSC was 1 mol, the dye labeling ratio (D/P) was 0.31, 0.58, 1. It was 2, 2.5. On the other hand, under the condition that the WSC is 3 times mol, the D/P is 0.81, 1.7, 2.8, and 4.7 when the amount of the dye charged is 2, 5, 10, and 20 times mol, respectively. there were. From this, it was confirmed that a larger amount of dye can be introduced into HSA when WSC is added in a 1-fold molar amount than in a 3-fold molar amount.

Claims (8)

And at least one organic dye is covalently bound contrast agent for photoacoustic imaging with a plurality of complexes comprising albumin, the organic dye is Ri compound or a salt thereof der represented by the following formula (1), said light A contrast agent for photoacoustic imaging, wherein the dye labeling rate in the contrast agent for acoustic imaging is 4 or more and 10 or less .

In the above formula (1), R _{1 to} R ₈ may independently be the same or different and each represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or a sulfonic acid group, and R _{9 to} R 8 ₁₂ may independently be the same or different and each represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, R ₁₃ and R ₁₄ may be the same or different and each have a substituent. Represents an optionally substituted alkyl group having 1 to 5 carbon atoms, the substituent is any one of a carboxyl group, a sulfonic acid group and a phosphonic acid group, n ₁ and n ₂ may be the same or different, 0 or 1, L _{1 to} L ₇ are methine groups, L _{1 to} L ₇ may form a 4-membered ring to a 6-membered ring, and R ₁₀₀ is a phenyl group, a pyridyl group or a substituent. Represents at least one of an alkyl group having 1 to 5 carbon atoms which may have, and the substituent is an aryl group, an arylalkyl group, a sulfoalkyl group, an alkylthio group, a sulfoalkylthio group, a complex ring group, an acylamino group. Group, an arylamino group, an N-aryl-N-alkylamino group, an arylthio group, an aryloxy group, a halogen atom, and an acylaminoaryloxy group. The contrast agent for photoacoustic imaging according to claim 1, wherein R ₁₀₀ is any of a methyl group, an ethyl group, a phenyl group, a pyridyl group, and a benzyl group. The contrast agent for photoacoustic imaging according to claim 1 or 2 , wherein the organic dye is a compound represented by any one of the following formulas (2) to (6) or a salt thereof.

In the above formulas (2) to (6), X is any one of a hydrogen atom, a sodium atom, a potassium atom and an iodine atom. The contrast agent for photoacoustic imaging according to any one of claims 1 to 3 , wherein the organic dye is a compound represented by the following formula (7) or a salt thereof.

The photoacoustic imaging according to any one of claims 1 to 4 , wherein the albumin is any one of human serum albumin, a modified form of human serum albumin, a fragment of human serum albumin, and a modified fragment of human serum albumin. Contrast agent. Contrast agent for photoacoustic imaging according to any one of claims 1 to 5 capturing molecule is bound to the albumin. Contrast agent for photoacoustic imaging according to any one of claims 1 to 6 is used to image the tumor. The contrast agent for photoacoustic imaging according to any one of claims 1 to 6 , which is used for imaging a lymph node.

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