WO2016143017A1 - Nanoparticule pour imagerie photo-acoustique - Google Patents

Nanoparticule pour imagerie photo-acoustique Download PDF

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WO2016143017A1
WO2016143017A1 PCT/JP2015/056754 JP2015056754W WO2016143017A1 WO 2016143017 A1 WO2016143017 A1 WO 2016143017A1 JP 2015056754 W JP2015056754 W JP 2015056754W WO 2016143017 A1 WO2016143017 A1 WO 2016143017A1
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infrared absorbing
polymer
photoacoustic imaging
nanoparticle
nanoparticles
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PCT/JP2015/056754
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Japanese (ja)
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英一 小関
功 原
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株式会社島津製作所
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Priority to PCT/JP2015/056754 priority Critical patent/WO2016143017A1/fr
Priority to TW105102762A priority patent/TW201632456A/zh
Publication of WO2016143017A1 publication Critical patent/WO2016143017A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo

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  • the present invention relates to nanoparticles used for photoacoustic imaging.
  • Patent Document 1 describes that an amphiphilic block polymer having a hydrophilic block chain containing a sarcosine unit and a hydrophobic block chain containing a lactic acid unit is useful as a probe agent for positron tomography or fluorescence imaging. Has been.
  • Biological imaging methods such as positron tomography (PET), fluorescence imaging (FI), nuclear magnetic resonance imaging (MRI), and ultrasound imaging (US) are methods for grasping the situation inside the living body without dissecting the living body. It has attracted attention in recent years.
  • PET positron tomography
  • FI fluorescence imaging
  • MRI nuclear magnetic resonance imaging
  • US ultrasound imaging
  • Photoacoustic imaging which is a kind of biological imaging method, is a method for obtaining an image to be measured by detecting the generation position and intensity of an acoustic wave (generally an ultrasonic wave) generated by the photoacoustic effect.
  • the photoacoustic effect is a phenomenon in which molecules that absorb light energy release heat, and an acoustic wave is generated by volume expansion due to the heat.
  • Photoacoustic imaging is a technique for detecting ultrasonic waves with low spatial resolution as an excitation means and detecting ultrasound with low attenuation in the tissue. It is attracting attention as.
  • a near-infrared (wavelength 700 to 900 nm) nanopulse laser with high biological permeability is used.
  • the target site may be difficult to observe due to near-infrared absorption by hemoglobin or melanin in vivo. Therefore, in order to effectively amplify the acoustic wave from the site to be measured and enable more detailed detection and measurement, a contrast agent having near infrared absorptivity such as gold nanoparticles and single-walled carbon nanotubes is used. Yes. However, since contrast agents made of inorganic materials are toxic, administration to living bodies is limited.
  • organic molecules such as indocyanine green (ICG) are known as contrast agents with low toxicity to living bodies.
  • ICG indocyanine green
  • near-infrared absorbing organic molecules such as ICG have a small molecular size and therefore have low accumulation at the site to be measured. Therefore, it has been proposed that a near infrared absorbing organic molecule such as ICG is encapsulated in a nanoparticle and used as a contrast agent for photoacoustic imaging.
  • Patent Document 2 discloses that particles in which ICG aggregates are encapsulated in liposomes can be used as a contrast agent for photoacoustic imaging.
  • Patent Document 2 discloses that a target site can be specifically labeled by immobilizing a substance (such as an antibody) that specifically binds to a target site such as a tumor on a particle.
  • Patent Document 2 only shows that liposomes encapsulating near-infrared absorbing dyes have a high retention rate in serum, and no examples of application to living bodies are shown. Moreover, the measurement example of photoacoustic imaging is not shown, and it cannot be said that the usefulness is shown.
  • an object of the present invention is to provide nanoparticles for photoacoustic imaging that use organic molecules that are less toxic to the living body and that are excellent in accumulation in the living body.
  • the present invention relates to nanoparticles for photoacoustic imaging used as a contrast agent for photoacoustic imaging.
  • the nanoparticle of the present invention comprises a molecular assembly including an amphiphilic block polymer having a hydrophilic block chain having 20 or more sarcosine units and a hydrophobic block chain having 10 or more lactic acid units.
  • the molecular assembly includes a near-infrared absorbing organic molecule containing a near-infrared absorbing functional group at a concentration of 5 ⁇ mol / g or more.
  • the particle diameter of the molecular assembly is preferably 20 to 200 nm.
  • the near-infrared absorbing organic molecule is a hydrophobic polymer in which a near-infrared absorbing functional group is bonded to a polymer chain containing 10 or more lactic acid units.
  • the near infrared absorbing functional group is, for example, a functional group represented by the following formula (I).
  • R 1 is an alkyl group which may be substituted
  • R 2 is an alkylene group which may be substituted
  • R 3 and R 3 ′ are a hydrogen atom or a group that is linked to each other to form a cyclic structure.
  • X is hydrogen or halogen.
  • Ring B and ring D are each a nitrogen-containing fused aromatic heterocyclic ring which may be the same or different.
  • a ⁇ is an anion
  • m is 0 or 1.
  • the near-infrared absorbing functional group is a group derived from indocyanine green.
  • the nanoparticle of the present invention is an organic molecule and excellent in biodegradability, it has low toxicity when administered to a living body.
  • the nanoparticle of the present invention has the property of specifically accumulating in vascular lesions and cancerous sites. Therefore, by applying the nanoparticles of the present invention as a contrast agent to a living body and performing photoacoustic imaging using near-infrared excitation light, the position of vascular lesions and cancerous sites can be visualized and accurately determined. Can do.
  • FIG. 1 It is a block diagram of the photoacoustic imaging apparatus used in the Example.
  • A is the absorption spectrum of the nanoparticle (ICG lactosome) of an Example
  • B is the absorption spectrum of ICG.
  • the photoacoustic imaging measurement results, (A) to (D) are the photoacoustic signals with the excitation light of 796 nm, and (E) and (F) are the visualization of the photoacoustic signals with the excitation light of 532 nm. .
  • a nanoparticle consists of a molecular assembly containing an amphiphilic block polymer, and includes a near-infrared absorbing organic molecule in the molecular assembly.
  • an amphiphilic block polymer forms a molecular assembly together with a near infrared absorbing polymer; an amphiphilic block polymer to which a near infrared absorbing group is bonded is a molecule.
  • amphiphilic block polymer In the nanoparticle of the present invention, the cohesive force of the amphiphilic block polymer becomes a driving force to form a molecular assembly. That is, the amphiphilic block polymer is a basic element of the molecular assembly.
  • the amphiphilic block polymer has a hydrophilic block chain and a hydrophobic block chain.
  • Hydrophilic of the hydrophilic block chain means that the hydrophilic block chain is relatively hydrophilic. Specifically, the hydrophilic block chain means hydrophilicity to such an extent that the copolymer molecule as a whole can realize amphiphilicity by forming a block copolymer with the hydrophobic block chain. Similarly, “hydrophobic” of the hydrophobic block chain means that the hydrophobic block chain is relatively hydrophobic with respect to the hydrophilic block chain. Specifically, this means that the hydrophobic block chain forms a block copolymer with the hydrophilic block chain so that the copolymer molecule as a whole can realize amphipathic properties.
  • the hydrophilic block chain is a hydrophilic molecular chain having 20 or more units (sarcosine units) derived from sarcosine (N-methylglycine). Sarcosine is highly water soluble.
  • polysarcosine since polysarcosine has an N-substituted amide, cis-trans isomerization is possible, and since there is little steric hindrance around the ⁇ -carbon, it has high flexibility. Therefore, by using a polysarcosine chain as a structural unit, a hydrophilic block chain having both high hydrophilicity and flexibility is formed.
  • the hydrophilic blocks of the adjacent block polymer are easily aggregated, and the self-aggregation property is enhanced, so that a molecular assembly is easily formed.
  • the upper limit of the number of sarcosine units in the hydrophilic block chain is not particularly limited, but is preferably 500 or less from the viewpoint of stabilizing the structure of the molecular assembly. If the number of sarcosine units is too large, the molecular assembly tends to lack stability.
  • the number of sarcosine units in the hydrophilic block chain is more preferably 30 to 300, and even more preferably 50 to 200.
  • all sarcosine units may be continuous, or the sarcosine units may be discontinuous as long as the properties of the above polysarcosine are not impaired.
  • the hydrophilic block chain has a monomer unit other than sarcosine, the monomer unit other than sarcosine is not particularly limited, and examples thereof include a hydrophilic amino acid or an amino acid derivative.
  • Amino acids include ⁇ -amino acids, ⁇ -amino acids, and ⁇ -amino acids, and are preferably ⁇ -amino acids.
  • hydrophilic ⁇ -amino acids include serine, threonine, lysine, aspartic acid, glutamic acid and the like.
  • the hydrophilic block may have a sugar chain, a polyether or the like.
  • the hydrophilic block preferably has a hydrophilic group such as a hydroxyl group at the terminal (terminal opposite to the linker part with the hydrophobic block).
  • the hydrophilic block chain may be a straight chain or may have a branched structure.
  • each branch chain contains two or more sarcosine units.
  • the hydrophobic block chain has 10 or more lactic acid units (in this specification, this hydrophobic block chain having a lactic acid unit as a basic unit may be simply referred to as polylactic acid).
  • Polylactic acid has excellent biocompatibility and stability. Therefore, a molecular assembly obtained from an amphiphilic polymer having polylactic acid as a building block is useful in applications to living bodies, particularly the human body.
  • polylactic acid since polylactic acid has excellent biodegradability, it is rapidly metabolized and has low accumulation in tissues other than the lesion site in vivo. Therefore, a molecular assembly obtained from an amphiphilic substance having polylactic acid as a building block is very useful in terms of specific accumulation in vascular lesions.
  • polylactic acid has high solubility in a low-boiling solvent
  • a low-boiling organic solvent can be used for the solution for producing the molecular assembly. Therefore, the remaining amount of the solvent used for the production of the molecular assembly can be easily reduced, and the safety to the living body is excellent, and the production efficiency of the molecular assembly is increased.
  • the hydrophobic block chain has 10 or more lactic acid units, a hydrophobic core is easily formed and the self-aggregation property is enhanced, so that a molecular assembly is easily formed.
  • the upper limit of the number of lactic acid units in the hydrophobic block chain is not particularly limited, but is preferably 100 or less from the viewpoint of stabilizing the structure of the molecular assembly.
  • the number of lactic acid units in the hydrophobic block is more preferably 20 to 80, and further preferably 30 to 50.
  • Adjustment of the chain length of polylactic acid is also preferable in that it contributes as a factor in shape control and size control of a molecular assembly obtained from an amphiphilic substance having polylactic acid as a building block.
  • all of the lactic acid units may be continuous or discontinuous.
  • the lactic acid unit constituting the hydrophobic block chain may be L-lactic acid or D-lactic acid. Further, L-lactic acid and D-lactic acid may be mixed. In the hydrophobic block chain, all lactic acid units may be continuous, or the lactic acid units may be discontinuous.
  • the monomer unit other than lactic acid contained in the hydrophobic block chain is not particularly limited, for example, hydroxy acid such as glycolic acid, hydroxyisobutyric acid, glycine, alanine, valine, leucine, isoleucine, proline, methionine, tyrosine, tryptophan,
  • hydroxy acid such as glycolic acid, hydroxyisobutyric acid, glycine, alanine, valine, leucine, isoleucine, proline, methionine, tyrosine, tryptophan
  • hydrophobic amino acids or amino acid derivatives such as glutamic acid methyl ester, glutamic acid benzyl ester, aspartic acid methyl ester, aspartic acid ethyl ester, and aspartic acid benzyl ester.
  • the hydrophobic block chain may be linear or may have a branched structure.
  • a compact hydrophobic core tends to be formed at the time of molecular assembly formation, and the density of the hydrophilic block chain tends to increase. Therefore, in order to form a core / shell type molecular assembly having a small particle size and high structural stability, the hydrophobic block chain is preferably linear.
  • the amphiphilic polymer is obtained by bonding a hydrophilic block chain and a hydrophobic block chain.
  • the hydrophilic block chain and the hydrophobic block chain may be bonded via a linker.
  • the linker includes a lactic acid monomer (lactic acid or lactide), which is a structural unit of a hydrophobic block chain, or a functional group (for example, a hydroxyl group, an amino group, etc.) capable of binding to a polylactic acid chain and a sarcosine, which is a structural unit of a hydrophilic block.
  • a monomer for example, sarcosine or N-carboxysarcosine anhydride
  • a functional group for example, an amino group
  • the method for synthesizing the amphiphilic block polymer is not particularly limited, and a known peptide synthesis method, polyester synthesis method, depsipeptide synthesis method, or the like can be used. Specifically, an amphiphilic block polymer can be synthesized with reference to WO 2009/148121 (Patent Document 1).
  • Near-infrared absorbing organic molecules contain functional groups that absorb near-infrared (wavelength 700-1300 nm). The substituent of the compound having a hydrogen bond has absorption in the near infrared region, but the absorption is relatively small, and the near infrared ray is likely to pass through living tissue. Therefore, if a contrast agent containing a near-infrared absorbing organic molecule that absorbs near-infrared rays and releases thermal energy is administered to a living body and photoacoustic imaging is performed, information on a site where the contrast agent is integrated can be obtained accurately. It becomes possible. That is, the inclusion of near-infrared absorbing organic molecules in the nanoparticles enables photoacoustic imaging of a living body using near-infrared excitation light.
  • Examples of the near-infrared absorbing organic molecule include those having a form in which a near-infrared absorbing group is bonded to a polymer (near-infrared absorbing polymer) and the near-infrared absorbing organic molecule itself (near-infrared absorbing agent).
  • the near-infrared absorbing polymer is a polymer in which a near-infrared absorbing group is bonded to a polymer chain.
  • a near-infrared absorbing group bonded to the above-mentioned amphiphilic polymer may be used as a near-infrared absorbing polymer, or a near-infrared absorbing group bonded to a polymer capable of forming a molecular assembly together with the amphiphilic polymer. Further, it may be used as a near infrared absorbing polymer.
  • near-infrared absorbing group those constituting a long conjugated ⁇ -electron system by combining a plurality of aromatic rings through a conjugated system are used.
  • Specific examples include functional groups derived from dyes such as cyanine dyes, phthalocyanine dyes, naphthoquinone dyes, diimmonium dyes, and azo dyes.
  • cyanine is preferable because it has a high near-infrared extinction coefficient and can easily increase the photoacoustic signal intensity.
  • An example of a group derived from a cyanine dye is represented by the following general formula (I).
  • R 1 is an alkyl group which may be substituted
  • R 2 is an alkylene group which may be substituted
  • R 3 and R 3 ′ are a hydrogen atom or a group that is linked to each other to form a cyclic structure.
  • X is hydrogen or halogen.
  • Ring B and ring D are each a nitrogen-containing fused aromatic heterocyclic ring which may be the same or different.
  • a ⁇ is an anion, and m is 0 or 1.
  • Ring B and ring D are nitrogen-containing fused aromatic heterocycles which may be the same or different.
  • R 1 (alkyl group) and R 2 (alkylene group) have 1 to 20 carbon atoms, preferably 2 to 5 carbon atoms.
  • the substituent may be anionic.
  • the substituent include a carboxyl group, a carboxylate group, a metal carboxylate group, a sulfonyl group, a sulfonate group, a metal sulfonate group, and a hydroxyl group.
  • the metal may be an alkali metal or an alkaline earth metal.
  • the halogen can be Cl, Br and I.
  • R 1 and R 2 are an anionic group and takes a betaine structure as a whole molecule.
  • a ⁇ can be a halogen ion such as Cl ⁇ , Br ⁇ and I ⁇ , CIO 4 ⁇ , BF 4 ⁇ , PF 6 ⁇ , SbF 6 ⁇ , SCN ⁇ and the like.
  • Ring A and Ring B may each independently be a nitrogen-containing bicyclic or tricyclic aromatic heterocycle.
  • ring B and ring D are the same.
  • ring B the following structures are exemplified.
  • the following structures are exemplified.
  • R 4 and R 5 can both be hydrogen.
  • R 4 and R 5 may be linked to each other to form an aryl ring.
  • the aryl ring may be a benzene ring that may be substituted.
  • a preferred near infrared absorbing group is a group derived from an indocyanine compound represented by the following structural formula (II).
  • preferred near-infrared absorbing groups include ICG group (formula III below), IC7-1 group (formula formula IV below) and IR820 group, wherein both ring B and ring D are nitrogen-containing tricyclic aromatic heterocycles (Formula V); IR783 group (Formula VI below) and IR806 group (Formula VII below) in which both ring B and ring D are nitrogen-containing bicyclic aromatic heterocycles; and ring B is a nitrogen-containing tricycle And an IC7-2 group (formula VIII below) wherein the ring C is a nitrogen-containing bicyclic aromatic heterocycle.
  • the structure of the polymer chain portion of the near infrared absorbing polymer is not particularly limited.
  • the polymer chain is preferably hydrophobic. If the near-infrared absorbing polymer is hydrophobic, it is easy to aggregate in the hydrophobic core of the molecular assembly, and nanoparticles with high photoacoustic signal intensity due to near-infrared excitation light can be obtained.
  • the hydrophobic polymer chain preferably has a plurality of lactic acid units.
  • the main component may be a lactic acid unit (ie, a polylactic acid chain), or may have a hydrophilic block in addition to the hydrophobic block of a plurality of lactic acid units (ie, an amphiphilic block polymer chain).
  • the hydrophobic polymer chain of the near-infrared absorbing polymer preferably has 5 or more lactic acid units.
  • the number of lactic acid units in the hydrophobic polymer chain is more preferably 5 to 50, and further preferably 15 to 35. All lactic acid units may be continuous or discontinuous.
  • the structural unit and chain length of the hydrophobic polymer chain of the near-infrared absorbing polymer are basically the hydrophobic block in the above-mentioned amphiphilic block polymer. It can be determined from the same viewpoint as the molecular design of the chain. In this way, since the affinity between the near-infrared absorbing polymer and the hydrophobic block chain of the amphiphilic block polymer is high, it is easy to obtain a molecular assembly including the near-infrared absorbing polymer in the hydrophobic core portion.
  • the polymer chain of the near infrared absorbing polymer does not exceed the length of the amphiphilic block polymer.
  • the polymer chain of the near infrared absorbing polymer preferably does not exceed twice the length of the hydrophobic block in the amphiphilic block polymer.
  • the near infrared absorbing group is preferably bonded to the terminal structural unit of the polymer chain.
  • n is an integer, preferably 5-50.
  • a near-infrared absorber can form a part of a molecular assembly by being encapsulated in the molecular assembly having the above-mentioned amphiphilic block polymer as a basic element.
  • the near infrared absorber include cyanine dyes, phthalocyanine dyes, naphthoquinone dyes, diimmonium dyes, azo dyes, and the like. Among these, cyanine is preferable because it has a high near-infrared extinction coefficient and can easily increase the photoacoustic signal intensity.
  • An example of a cyanine dye is represented by the following general formula (I ′).
  • R 2 ′ is obtained by adding a hydrogen atom, an alkyl group, or other substituents to R 2 in the above formula (I).
  • the formula (I ') is, R 2 is R 2' except that replaced, the same as in the above formula (I).
  • the near-infrared absorber represented by the above formula (I ′) include ICG (formula III ′ below), IC7-1, wherein both ring B and ring D are nitrogen-containing tricyclic aromatic heterocycles (Formula IV 'below) and IR820 (Formula V' below); IR783 (Formula VI 'below) and IR806 (Formula VII' below) where both ring B and ring D are nitrogen-containing bicyclic aromatic heterocycles; And IC7-2 (formula VIII ′ below), wherein ring B is a nitrogen-containing tricyclic aromatic heterocycle and ring C is a nitrogen-containing bicyclic aromatic heterocycle.
  • the nanoparticle of the present invention comprises a molecular assembly of the amphiphilic polymer formed so as to include the above-mentioned near-infrared absorbing organic molecule.
  • the nanoparticle containing a near-infrared absorption organic molecule is obtained by forming the molecular assembly of an amphiphilic block polymer.
  • the method for producing the nanoparticles is not particularly limited, and can be appropriately selected according to the size and characteristics of the nanoparticles, the type, properties, content, etc. of the near-infrared absorbing organic molecules to be supported. The formation of nanoparticles can be confirmed by observation with an electron microscope.
  • a film obtained by drying a solution containing an amphiphilic polymer and a near-infrared absorbing organic molecule is brought into contact with an aqueous liquid, and the nanoparticles are dispersed in the aqueous liquid.
  • a method for obtaining a dispersion film method
  • a method for obtaining a dispersion by bringing the solution into contact with an aqueous liquid injection method.
  • a solution containing a component of a molecular assembly such as an amphiphilic block polymer or a near infrared absorbing organic molecule and a solvent is used.
  • the solvent is not particularly limited as long as it can dissolve the components of the molecular assembly.
  • a low boiling point solvent is preferably used.
  • the low boiling point solvent means a solvent having a boiling point at 1 atm of 100 ° C. or lower, preferably 90 ° C. or lower.
  • the solution used for forming the nanoparticles may contain components other than the amphiphilic block polymer, the near-infrared absorbing organic molecule, and the solvent.
  • a polymer other than the amphiphilic block polymer or near-infrared absorbing polymer may be included as a component of the molecular assembly.
  • the polymer other than the amphiphilic block polymer and the near infrared absorbing polymer include hydrophobic polymers.
  • hydrophobic polymer a polymer equivalent to the polymer chain of the near-infrared absorbing polymer (not including a near-infrared absorbing group) can be used.
  • hydrophobic polymers other than near-infrared absorbing polymers ie, hydrophobic polymers that do not contain near-infrared absorbing groups. Or it is preferable to reduce the amount used.
  • the amount of the hydrophobic polymer containing no near infrared absorbing group is preferably 30 parts by mole or less, more preferably 20 parts by mole or less, and more preferably 10 parts by mole or less with respect to 100 parts by mole of the near infrared absorbing polymer. Further preferred.
  • the film method is a method used for preparing liposomes. Since the above-mentioned amphiphilic block polymer has a hydrophobic block chain having a lactic acid unit and is soluble in a low-boiling solvent, nanoparticles can be prepared using this method.
  • a step of preparing the above solution in a container such as a glass container; a step of removing an organic solvent from the solution and obtaining a film containing a polymer and a near infrared absorbing organic molecule on the inner wall of the container; and in the container Adding a water-based liquid to the film, and converting the film-like substance into a molecular assembly containing near-infrared absorbing organic molecules to obtain a dispersion of nanoparticles.
  • a film containing a polymer constituting the molecular assembly and near-infrared absorbing organic molecules is formed on the inner wall of the container.
  • the method for removing the solvent is not particularly limited, and can be appropriately determined according to the boiling point of the solvent to be used.
  • the solvent may be removed under reduced pressure, or the solvent may be removed by natural drying.
  • a molecular assembly is formed in the process in which an aqueous liquid is added to the container to which this film is attached and the film is peeled off from the inner wall of the container.
  • the aqueous liquid is water or an aqueous solution, and may be any one that is biochemically and pharmaceutically acceptable, and examples thereof include distilled water for injection, physiological saline, and buffer solution.
  • a heating treatment or an ultrasonic treatment may be performed after the aqueous liquid is added to the container.
  • the heating treatment can be performed, for example, under conditions of 70 to 100 ° C. and 5 to 60 minutes.
  • a film may be formed on a substrate such as a film or a glass plate, and nanoparticles may be formed by contacting the film on the substrate with an aqueous liquid. It can.
  • the contact between the film on the substrate and the aqueous liquid can be performed, for example, by immersing the substrate on which the film is formed in the aqueous liquid.
  • the injection method is a method used for the preparation of many other nanoparticles.
  • nanoparticles are prepared by dispersing the above solution in an aqueous liquid and performing purification treatment such as gel filtration chromatography, filtering, ultracentrifugation, etc., and then removing the organic solvent. Can do.
  • purification treatment such as gel filtration chromatography, filtering, ultracentrifugation, etc.
  • the nanoparticle dispersion obtained by the film method or the injection method can be directly administered to a living body as a contrast agent for photoacoustic imaging. Further, the obtained dispersion liquid may be freeze-dried. As a freeze-drying treatment method, a known method can be adopted. For example, the nanoparticle dispersion liquid is frozen with liquid nitrogen or the like and sublimated under reduced pressure to obtain a freeze-dried nanoparticle product. This makes it possible to store the nanoparticles as a lyophilized product. If necessary, nanoparticles can be used for use by adding an aqueous liquid to the lyophilized product to obtain a dispersion of nanoparticles.
  • the dispersion before the lyophilization treatment there may be a case where a polymer or a near infrared absorbing organic molecule that has not contributed to the formation of nanoparticles remains as such.
  • a dispersion is subjected to a freeze-drying process, it is possible to form nanoparticles further from the polymer and the near-infrared absorbing organic molecules that remain without forming nanoparticles during the process of solvent concentration. become. Therefore, it is possible to efficiently prepare the nanoparticles of the present invention.
  • concentration of the near-infrared absorption organic molecule in the nanoparticle for photoacoustic imaging is 5 micromol / g or more.
  • concentration of the near-infrared absorbing organic molecules By increasing the concentration of the near-infrared absorbing organic molecules, the intensity of the photoacoustic signal when nanoparticles are administered to a living body can be increased.
  • concentration of the near-infrared absorbing organic molecule is less than 5 ⁇ mol / g, it is difficult to obtain a photoacoustic signal to the extent that living body imaging can be performed even if the dose of nanoparticles to the living body is increased.
  • the concentration of the near-infrared absorbing organic molecule in the nanoparticles is preferably 10 ⁇ mol / g or more, more preferably 15 ⁇ mol / g or more, and further preferably 20 ⁇ mol / g or more.
  • the upper limit of the concentration of the near-infrared absorbing organic molecules in the nanoparticles is not particularly limited, and the photoacoustic signal intensity tends to increase as the concentration increases. However, if the concentration of the near-infrared absorbing organic molecule is too high, the structure of the molecular assembly tends to become unstable.
  • the concentration of the near-infrared absorbing organic molecule in the nanoparticles is preferably 100 ⁇ mol / g or less, more preferably 70 ⁇ mol / g or less, and further preferably 50 ⁇ mol / g or less.
  • Near-infrared absorbing groups such as ICG are also fluorescent dyes and emit fluorescence when irradiated with near-infrared rays, and are therefore used as fluorescent contrast agents.
  • fluorescence imaging when the concentration of the fluorescent dye increases, the signal decreases due to concentration quenching.
  • concentration of the fluorescent dye molecules is in close proximity. Quenching easily occurs. For example, when ICG is encapsulated in a nanoparticle of a molecular assembly, when the concentration is 5 ⁇ mol / g or more, fluorescence from ICG is hardly observed.
  • the concentration of the near-infrared absorbing organic molecules in the nanoparticles can be increased by adjusting the use ratio of the amphiphilic polymer and the near-infrared absorbing organic molecules.
  • the amount of the near-infrared absorbing polymer is 5 with respect to 100 mol parts of the amphiphilic polymer. ⁇ 200 mol parts are preferable, 10 to 100 mol parts are more preferable, and 15 to 70 mol parts are more preferable.
  • the size of the nanoparticle for photoacoustic imaging of the present invention is preferably 20 to 200 nm.
  • “Particle size” refers to the particle size that appears most frequently in the particle distribution, that is, the center particle size.
  • the size of the nanoparticles can be measured by an observation method using a transmission electron microscope (TEM) or a dynamic light scattering (DLS) method.
  • the method for controlling the size of the nanoparticles includes a method for adjusting the chain length of the amphiphilic block polymer and the hydrophobic polymer, and a method for adjusting the blending amount of the hydrophobic polymer. As the blending amount of the hydrophobic polymer increases, the volume of the hydrophobic core of the molecular assembly increases, so that the nanoparticles tend to increase.
  • the nanoparticle for photoacoustic imaging of the present invention is a molecular assembly (so-called lactosome) having an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit as basic elements.
  • lactosome has a feature that it has a high blood retention and a remarkably low accumulation amount in the liver as compared with other molecular assemblies.
  • pigments such as near-infrared absorbers can be used for photoacoustic imaging.
  • pigments such as indocyanine green have high accumulation in the liver, and lesions such as blood vessels are present at the lesion site. It was difficult to accumulate at the site.
  • the nanoparticle of the present invention if used, the property that the nanoparticles staying in the blood easily accumulate in a vascular lesion site such as a tumor site, an inflammatory site, an arteriosclerosis site, or an angiogenesis site ( By utilizing the EPR (enhanced “permeability” and “retention” effect), photoacoustic imaging targeting a cancerous part or the like becomes possible.
  • the living body to which the nanoparticles are administered is not particularly limited, and may be a human or non-human animal.
  • Non-human animals include mammals other than humans, more specifically primates, rodents (mouse, rat, etc.), rabbits, dogs, cats, pigs, cows, sheep, horses and the like.
  • the method of administration into the living body is not particularly limited, and any of systemic administration and local administration may be used. That is, administration of nanoparticles can be performed by any of injection, internal use, and external use.
  • the concentration of nanoparticles in the administration liquid is about 0.5 to 100 mg / mL, preferably 0.8 to 50 mg / mL, more preferably 1 to 20 mg / mL.
  • the time from administration of the nanoparticle to the start of detection can be appropriately determined according to the type of near-infrared absorbing organic molecule that the nanoparticle has, the type of administration target, and the like. For example, it can be 3 to 48 hours after administration, or 1 to 24 hours. Below the above range, if the nanoparticles are not sufficiently retained in the target, such as a cancerous site, or if the signal is too strong to clearly separate the administration target from other sites (background) There is. When the above range is exceeded, nanoparticles may be excreted from the administration target.
  • Photoacoustic imaging device Photoacoustic imaging uses a phenomenon (photoacoustic effect) in which a molecule that absorbs light energy releases heat and an acoustic wave is generated by volume expansion due to the heat.
  • an appropriate apparatus can be used according to the imaging region or the like.
  • the photoacoustic imaging apparatus includes a photoexcitation unit, an acoustic wave detection unit, and an imaging unit.
  • the photoexcitation unit irradiates near-infrared rays to excite near-infrared absorbing organic molecules contained in nanoparticles administered to the living body.
  • a near-infrared coherent light source is preferable, and among them, an OPD laser is preferably used.
  • the OPD laser is a coherent light source based on optical parametric oscillation (Optical Parametric Oscillation; OPD), and has been put into practical use as a light source having a continuously variable wavelength in a wide wavelength range from the visible range to the infrared range.
  • the acoustic wave detection unit detects an acoustic wave (specifically, an ultrasonic wave) generated by volume expansion due to heat released from the molecules excited by the irradiation light from the photoexcitation unit.
  • an acoustic sensor capable of detecting ultrasonic waves can be used.
  • the imaging unit only needs to be able to visualize the position and size of the tissue where the nanoparticles are present based on the position of the acoustic wave detection unit and the waveform of the detected acoustic wave. From the obtained photoacoustic image, it is possible to know the staying state of the nanoparticles in the living body, and based on this, it is possible to diagnose the presence / absence or position of a vascular lesion.
  • Nanoparticles of a molecular assembly composed of a near-infrared absorbing polymer (ICG-PLLA 30 ) and an amphiphilic block polymer (PSar 70 -PLLA 30 ) were prepared as follows.
  • glycolic acid 72 mg, 0.95 mmol
  • O- (benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphoric acid Salt HATU
  • DIEA N, N-diisopropylethylamine
  • ICG lactosome The content of ICG-modified poly L-lactic acid in the nanoparticles was 27 ⁇ mol / g.
  • FIG. 2B shows an absorption spectrum of indocyanine green (concentration: 2 ⁇ M).
  • ICG lactosome had a smaller absorption peak width than that of ICG alone, and the molar extinction coefficient increased by about 10%.
  • the photoacoustic signal is proportional to the extinction coefficient. Therefore, it can be seen that by using the nanoparticles of the present invention, optical imaging with high signal intensity is possible compared to the case where the dye is used alone. The reason for the change in the maximum of the near-infrared absorption peak and the shape is not clear. However, in the nanoparticle of the present invention, since a plurality of ICG groups are closely fixed to the rigid body in one molecular assembly, the ICG It is considered that intermolecular interaction between groups occurs and the conjugated ⁇ -electron system is perturbed.
  • FIG. 1 is a schematic configuration diagram of the photoacoustic imaging measurement system used in this embodiment.
  • the photoacoustic excitation light source 1 a 30 Hz optical parametric oscillator (pulse width 6 nanoseconds) was used.
  • the excitation light source was adjusted to 796 nm corresponding to the absorption peak of ICG-lactosome.
  • the output light pulse is divided into four beams by the beam splitter 5, and each beam is guided to the optical fiber 11 through the lens 7.
  • the energy of the irradiation pulse light from each optical fiber was adjusted to less than 20 ⁇ J, and the intensity on the tissue surface was about 13 mJ / cm 2 .
  • the tips of the four optical fibers 11 are attached to the photoacoustic sensor head 15.
  • the imaging probe of the photoacoustic sensor head 15 has four excitation optical fibers 11 arranged obliquely and a 50 MHz ultrasonic sensor for acoustic wave detection provided with an acoustic lens. This probe has a depth resolution of 39 ⁇ m and an XY plane resolution of 56 ⁇ m.
  • the photoacoustic sensor head 15 is connected to the movable stage 13 and its position is controlled by the computer 50. In this example, a 4 mm square area was scanned in 150 ⁇ m steps. Photoacoustic waves (ultrasonic waves) induced by the optical pulses of the four excitation optical fibers 11 are amplified by the field effect transistor amplifier 25. The phototube 23 detects laser oscillation from the photoacoustic excitation light source 1, and using this as a trigger, the acoustic wave amplified by the amplifier 25 is detected and recorded by the digital oscilloscope 21 and averaged.
  • the time waveform of the photoacoustic wave is converted into a depth profile based on the speed of sound in the tissue (1540 m / sec), and reconstructed into a three-dimensional photoacoustic image by visualization software (Kitware VolView 3.4).
  • Photodynamic therapy was performed 18 hours after administration using a 808 nm fiber coupled CW laser diode. Laser irradiation was performed for 10 minutes (360 J / cm 2 ) at a spot size of 10 mm and a power density of 600 mW / cm 2 so as to cover the entire tumor region. A photoacoustic imaging image measured after PDT is shown in FIG.
  • Photoacoustic imaging of blood vessels Photoacoustic imaging measurements of blood vessels were performed before administration of ICG lactosomes and after PDT.
  • a 100 Hz optical parametric oscillator pulse width 9 nanoseconds
  • the excitation wavelength is adjusted to 532 nm, which is the isosbestic point of oxygenated hemoglobin (HbO 2 ) and deoxygenated hemoglobin (HHb). did.
  • Photoacoustic imaging images before administration and after PDT are shown in FIGS. 3 (E) and 3 (F).
  • the nanoparticle of the present invention was specifically accumulated in the blood vessel at the lesion site, and the concentration was rapidly decreased by PDT, which could be observed by photoacoustic imaging using near infrared rays. Yes. From these results, it can be seen that the nanoparticles of the present invention are useful for diagnosis of vascular lesions.

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Abstract

La nanoparticule de la présente invention comprend un agrégat moléculaire contenant un polymère à blocs amphiphiles. Le polymère à blocs amphiphiles comprend une chaîne de type bloc hydrophile constituée de 20 ou plusieurs unités sarcosine et une chaîne de type bloc hydrophobe constituée de 10 ou plus unités d'acide lactique. L'agrégat moléculaire contient une molécule organique absorbant le proche infrarouge, possédant un groupe fonctionnel absorbant le proche infrarouge, à une concentration de 5 µmoL/g ou plus. Il est préférable que la molécule organique absorbant l'infrarouge proche soit un polymère hydrophobe, au sein duquel ledit groupe fonctionnel absorbant le proche infrarouge est fixé à une chaîne polymère constituée de 10 ou plus unités d'acide lactique. La nanoparticule de la présente invention est utilisable en tant qu'agent de contraste pour l'imagerie photo-acoustique.
PCT/JP2015/056754 2015-03-06 2015-03-06 Nanoparticule pour imagerie photo-acoustique WO2016143017A1 (fr)

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WO2012128326A1 (fr) * 2011-03-23 2012-09-27 国立大学法人筑波大学 Nanoparticules pour une thérapie photodynamique
JP2014129318A (ja) * 2012-02-23 2014-07-10 Canon Inc インドシアニングリーン含有粒子、およびそれを有する光音響イメージング用造影剤
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* Cited by examiner, † Cited by third party
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US10828380B2 (en) 2015-09-30 2020-11-10 Canon Kabushiki Kaisha Conjugate of polysarcosine and NIR contrast agent for photoacoustic imaging

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