JP2019182778A - Contrast medium allowing both ultrasound imaging and near-infrared fluorescent imaging - Google Patents

Abstract

To observe a relatively wide range of shallow region with near-infrared fluorescent imaging, locate a site of interest (i.e., navigation), and then use ultrasound imaging to observe a deeper region with high resolution.SOLUTION: The contrast medium allowing both ultrasound imaging and near-infrared fluorescent imaging, comprises: an ultrasound contrast medium (A) comprising liposome particles (1) as a dispersoid and physiological saline solution (4) as a dispersion medium, the liposome particles having nano- or micro-sized diameter and containing a gas (2) therein; and a near-infrared fluorescent imaging medium (B) comprising a near infrared fluorescent dye (3); the liposome particles (1) and the near infrared fluorescent dye (3) being bonded to each other.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a contrast agent capable of both ultrasonic contrast and near infrared fluorescence contrast.

  In ultrasound contrast imaging, an ultrasound pulse that travels in a living body is reflected at a place where the living tissue changes (more precisely, where the acoustic impedance changes), and the reflected wave is processed and imaged (imaging). In order to enhance reflection, a contrast agent is used. A contrast agent is administered to a living tissue (for example, blood vessels or lymphatic vessels) to enhance reflection, thereby obtaining a new biological tissue image (for example, blood vessel or lymphatic distribution image) that has not been obtained so far. . Unlike X-rays, ultrasound has the advantage that there is no or very little damage to living tissue.

  Recently, if the size of bubbles (bubbles reflect ultrasound waves), which is the main role of ultrasound contrast media, is the size of micro or nano, the ultrasound contrast media depends on the size of living tissue (eg, liver, kidney, pancreas) , Deep in the gallbladder, stomach, stomach, duodenum, skin, etc. (specifically, when imaging from the skin surface, maximum depth: tens of mm, fan-shaped when imaging from the surface of organs such as the liver and heart) However, it is attracting attention because it reaches even the microvascular system and lymphatic system with a maximum depth of about 100 mm (see Non-Patent Documents 1 and 2).

  On the other hand, in the near-infrared light contrast method, a near-infrared fluorescent contrast agent is similarly administered to a living tissue, and near-infrared excitation light is irradiated from the outside. The contrast agent that has received the excitation light emits near-infrared light having a wavelength different from that of the excitation light when returning to the original ground state after being excited. Near-infrared light is relatively thick (specifically, a maximum thickness of about 10 mm) and transmits through living tissue, so that irradiation and reflected light or transmitted light can be received from the outside.

  Therefore, since the situation of a living tissue can be found with minimal invasiveness by photographing light emitted from the outside using a near-infrared light camera, it is widely used as a pathological diagnosis method for living tissue (affected part).

  Recently, a contrast agent is administered to blood vessels and lymph vessels of biological tissues (for example, liver, kidney, pancreas, gallbladder, stomach, stomach, duodenum, skin, etc.) and the depth (specifically, the maximum depth is 10 mm). (Non-Patent Documents 3 and 4) are attracting attention.

  The liposome of Non-Patent Document 4 is a liposome bound to indocyanine green (ICG), which is a near-infrared fluorescent contrast pigment that is the main role of a near-infrared fluorescent contrast agent, or an ICG derivative labeled with an alkyl chain. Yes, since it does not contain gas inside the liposome, it is not an ultrasound contrast agent but a near infrared fluorescent contrast agent. Liposomes are bound for the purpose of enlarging the dye molecules and causing the dye to stay and accumulate in the lymph nodes. After all, the liposome of Non-Patent Document 4 is completely different from the present invention regardless of ultrasound contrast.

  The “liposome containing fluorescent dye” in Patent Document 1 is the same as the liposome in Non-Patent Document 4. Basically, the liposome of Patent Document 1 is also a near-infrared fluorescent contrast agent, which is completely different from the present invention regardless of ultrasonic contrast.

  The liposome complex of Patent Document 2 is also a liposome particle to which indocyanine green (ICG) or the like is structurally bound, but contains a drug in the liposome particle. By irradiating the complex with a strong near infrared ray and causing the ICG to absorb it and generate heat, the liposome is destroyed and the drug is thereby released. It can also be said to be a drug used in a drug delivery system (DDS). After all, the liposome complex of Patent Document 2 is also a kind of near-infrared fluorescent contrast agent, which is completely different from the present invention regardless of ultrasonic contrast.

JP 2017-75937 A Republished 2013/051732

Y, Matsumura et al., Cancer research, 1986; 46: 6378-6392 Junichi Hirata et al., Drug Delivery System 2010; 25-5: 466-473 JT. Alander et al., International Journal of Biomedical Imaging, 2012; Article ID 940585: 26 Toyota et al., Bioorganic & Medicinal Chemistry 2014; 22: 721-727

  In the ultrasonic contrast method, a deep region is locally, that is, a relatively narrow range (width: several tens of millimeters from the body surface) and a relatively high resolution (depending on the frequency of the ultrasound, resolution: approximately several hundred μm). It is difficult to see a wide range (cons). In contrast, near-infrared fluorescence contrast imaging can observe a shallow region in a relatively wide range (width: several hundred mm) with high resolution (resolution: approximately several tens to several hundreds μm). The scope of application is narrow (cons).

  Therefore, the present inventors complemented the disadvantages of both, observed a relatively wide range of shallow region by near infrared fluorescence imaging, found the local to be seen (so to navigate), at the same position, This time, I thought that a new diagnostic method would be possible if the deep region could be observed locally and with high resolution by ultrasonic contrast.

  In order to make this idea feasible, it is necessary to devise both contrast agents. This is because the near-infrared fluorescent contrast agent diffuses relatively quickly, making it difficult to find the local area to be seen even if an ultrasound contrast agent is injected into the same region, and two types of injections are required in the same region. Therefore, there are problems such as that it takes a lot of time and effort and is likely to cause an error, and the management and safety risks of preparing two types of contrast agents are increased.

  In order to solve such a problem, the present inventors have conceived for the first time in the world that an ultrasonic contrast agent and a near-infrared fluorescent contrast agent are combined into one contrast agent. It came to be accomplished.

  Some ultrasonic contrast agents are mainly composed of liposomes (particles). Such a contrast agent is a dispersion (suspension) composed of liposome particles as a dispersoid and physiological saline as a dispersion medium, and contains a gas inside the liposome particles.

  Liposomes (particles) are artificially made by imitating a lipid bilayer membrane of a cell membrane, and are composed of a phospholipid film and an inclusion substance (described later).

  Phospholipids are amphipathic molecules that have a hydrophilic site (phosphocholine, etc., shown by ○ in FIG. 1) and a hydrophobic site (acyl group, shown by = in FIG. 1) containing a phosphorus atom. . When the phospholipid molecule is above a certain concentration in water (dispersion medium), the membranes spontaneously associate (merge) so that the hydrophilic sites are close to each other and the hydrophobic sites are close to each other. The film is formed into a spherical shape to minimize and stabilize the surface area. When an ultrasound contrast gas coexists, the gas is taken into the sphere because it is hydrophobic. This is what is called a “microemulsion” or “macroemulsion”. At this time, the phospholipid forms a monolayer.

  In the absence of gas, the phospholipid membrane is stabilized so that the two are close together on the front and back, and the membrane has a double structure, forming liposome particles. Since the inside of the liposome particles is also hydrophilic, water or an aqueous solution enters here.

  Depending on the method of preparing liposome particles into which a gas for ultrasound contrast is taken in, liposome particles in which a part of a sphere is a monolayer film and the other part is a bilayer film can be produced. In this case, gas is confined in the space surrounded by the single layer film, and water or an aqueous solution enters the remaining space.

  The present invention succeeded in mixing the liposome with a near-infrared fluorescent dye, which is the main component of the near-infrared fluorescent contrast agent, and binding it to a monolayer film or a bilayer film (liposome particles) during or after the liposome production. It came to be accomplished.

That is, the present invention
“An ultrasonic contrast agent (A) comprising a liposome particle as a dispersoid and a physiological saline as a dispersion medium, the diameter of the liposome particle being nano-sized or micro-sized, and containing a gas inside the liposome particle. A near-infrared fluorescent contrast agent (B) comprising an infrared fluorescent dye,
Contrast agent capable of both ultrasonic contrast and near-infrared fluorescence contrast, wherein the liposome particles and the near-infrared fluorescent dye are bonded "
I will provide a.

  Hereinafter, the contrast agent capable of both ultrasonic contrast and near-infrared fluorescence contrast according to the present invention may be referred to as a “dual contrast agent”.

  Generally speaking, the contrast agent of the present invention is a suspension composed of liposome particles as a dispersoid and physiological saline as a dispersion medium.

  The present invention provides the world's first contrast agent capable of both ultrasonic contrast and near infrared fluorescence contrast.

  The contrast agent of the present invention is a contrast agent capable of both ultrasonic contrast and near-infrared fluorescence contrast, and can reach both a shallow region and a deep region with a single injection, and the agent is relatively large at the molecular level. Since it has liposome particles, diffusion is slow even during near-infrared fluorescence imaging, and a clear image can be seen over time. Therefore, it is possible to discover a local area to be viewed over time and observe a deep region locally and with high resolution at the same position by ultrasonic contrast. Therefore, a new diagnostic method can be provided.

  Therefore, since it is not necessary to prepare two kinds of agents and only one kind is necessary, the above-described diagnostic method can be carried out by a single injection. Risk can be reduced.

  The contrast agent of the present invention can be broadly classified into two types according to the particle size of the liposome: nanometer (nm) size or micrometer (μm) size.

  In the case of nanometer (nm) size, it passes through the tissue gap and is absorbed into the lymphatic vessels. From these characteristics, it is possible to visualize the lymph flow from the administration by subcutaneous injection to the lymphatic vessels and lymph nodes, and it is expected to greatly contribute to the functional diagnosis of the lymphatic system near the body surface and the organ surface.

  In the case of a micrometer (μm) size, blood vessel-specific imaging can be performed by utilizing the characteristic that the tissue cannot pass through the tissue gap or the blood vessel wall. It can be administered intravenously and used to image blood vessels in various organs. As a main application, it is expected to be used as a surgical navigation technique for three-dimensionally grasping the blood vessel position in a surgical operation. In contrast, ultrasonic imaging uses a technique called Kupper imaging that uses the effect of macrophages on cells to image a tumor, but the same applies to near-infrared fluorescence imaging by using the contrast agent developed in the present invention. It is expected to perform imaging.

  Further, if the drug is encapsulated inside the liposome of the dual-use contrast agent of the present invention, the pharmacokinetic monitoring of the drug delivery system (DDS) can be performed by near-infrared fluorescence. After confirming that it has reached cancer), the drug is released by irradiating powerful ultrasound to destroy the liposome (the liposome collapses due to resonance vibration of the internal gas excited by the ultrasound) It becomes possible to administer the drug directly only to the affected part (for example, cancer).

It is an image figure of the contrast agent (liposome particle) of Example 1 (nanometer size). 3 is a graph showing the particle size distribution of the contrast agent of Example 1. It is the conceptual diagram which looked at the evaluation apparatus used by evaluation of the contrast agent of Example 1 from the side. When Example 1 and Comparative Example 1 were circulated, the fluorescence images of the channels observed and photographed from above using a near-infrared fluorescence observation apparatus [FIGS. 4 (1), (2), (5), (6 )] And tomographic images of the flow path [FIGS. 4 (3), (4), (7), (8)] taken by the ultrasonic diagnostic imaging apparatus. (1) is an appearance photograph of a pig foot, (2) an image of near-infrared fluorescence contrast, (3) is an appearance photograph of a lymph node part of the pig foot of (1), and (4) is near-infrared fluorescence. It is a fluorescence image of contrast. It is an ultrasound contrast image. Note that (1) is an image before contrast medium injection. It is an image figure of the contrast agent (liposome particle) of Example 2 (micrometer size). (1) is a photograph of the simplified flow path system of FIG. 3 (side view) taken from above, (2) and (3) are Example 2, Comparative Example 2 and PBS (saline) in the same system. FIG. 4 is a fluorescence image of the flow channel when the system is circulated and the system (FIG. 3 = side view) is observed and photographed from above, and (4) is a cut obtained by cutting the simple flow channel system of FIG. 3 (side view) vertically. It is an image figure of a surface. (5) and (6) are tomographic images of the flow path taken by the ultrasonic diagnostic imaging apparatus, and are the same plane as (4). It is an illustration which shows a mode that a contrast medium is injected into a chicken breast. (1) is an appearance photograph of the chicken breast, and (2) is a fluorescent image of the chicken breast. (3) is a tomographic image of the same breast breast by ultrasound contrast (before contrast medium administration), and (4) is a tomographic image of the same breast breast by ultrasound contrast (before contrast medium administration). It is the bright field image (photograph) observed using the optical microscope. It is the fluorescence image (photograph) acquired in fluorescence observation mode. It is a graph which shows an average particle diameter, particle concentration (number density), and particle size distribution. It is a molecular formula of near infrared fluorescence contrast. ICG-C18 is, R is _C 18 _{H 37.}

  Liposome-based ultrasound contrast agents that constitute contrast agents capable of both ultrasound contrast and near-infrared fluorescence contrast (both-purpose contrast agents) are already known per se, and the raw material of liposomes (particles) (phospholipids) , Lipids modified with polyethylene glycol (PEG-lipid, etc.) are readily available, and devices for preparing liposomes are also commercially available. In the contrast agent of the present invention, dispersoid liposomes (particles) are dispersed in physiological saline as a dispersion medium, and the whole image is a dispersion (suspension). The liposome is composed of a phospholipid film and an inclusion substance.

  Examples of the phospholipid constituting the film include phosphatidylcholine (DSPC), dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleyl phosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine ( DSPE, phosphatidylethanolamines such as diarachidoylphosphatidylethanolamine (DAPE) or dilinoleylphosphatidylethanolamine (DLPE); dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC) , Diarachidoyl-phosphatidylcholine (DAPC), dioleyl-phosphatidylcholine (DOPC), etc. Phosphatidylcholines of dimyristoylphosphatidylserine (DMPS), diarachidoylphosphatidylserine (DAPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleylphosphatidylserine (DOPS), etc .; dipalmitoyl Phophatidic acid derivatives such as phosphatidic acid (DPPA), dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), diarachidoyl phosphatidic acid (DAPA) and alkali metal salts thereof; dimyristoyl phosphatidylglycerol (DMPG) and its Alkali metal salt, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salt, distearoylphosphatidylglycero (DSPG) and its alkali metal salts, phosphatidylglycerols such as dioleyl-phosphatidylglycerol (DOPG); dilauroylphosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidyl Examples include phosphatidylinositol, cardiolipin, sphingomyelin lipid such as inositol (DPPI), distearoyl phosphatidylinositol (DSPI), dioleyl phosphatidylinositol (DOPI), and PEG-lipid as a PEG-lipid for increasing hydrophilicity. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [cyanur (modified with polyethylene glycol (Ethylene glycol) -2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [cyanur (polyethylene glycol) -2000, abbreviated as “DSPE-PEG2000”), stearyl PEG, palmitoyl PEG, oleic acid PEG, Examples include myristyl PEG and lauroyl PEG (the number following PEG indicates the molecular weight of the PEG moiety). The molecular weight of PEG is preferably 500 to 12,000. DSPE-PEG such as DSPE-PEG2000, DSPE-PEG3000, DSPE-PEG5000 is preferred. Of these, DSPE-PEG2000 is particularly preferable.

  Next, the inclusion substance will be described. Examples of the gas encapsulated in the liposome particles include air, oxygen, carbon dioxide, sulfur fluoride gas, and perfluorohydrocarbon gas. Examples of the perfluorohydrocarbon gas include perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, and the like.

  As a method of encapsulating gas, bubble liposomes encapsulating the gas can be prepared by placing liposome particles in a container filled with the gas and performing ultrasonic treatment or the like.

  On the other hand, examples of the near-infrared fluorescent dye that is the main component of the near-infrared fluorescent contrast agent include indocyanine green (ICG), phthalocyanine, squarylium, croconium, and derivatives thereof (ICG-C18).

  The wavelength of the excitation light of the near-infrared fluorescent dye is usually 600 nm or more, preferably 650 nm or more, more preferably 665 nm or more, further preferably 680 nm or more, and particularly preferably 700 nm or more. Most preferably, it is 720 nm or more. The fluorescence wavelength is preferably 50 nm or more, particularly 100 to 200 nm longer than the wavelength of the excitation light.

  The composition of the dual-use contrast agent of the present invention includes, as essential components, the above-described liposome particles, gas and near-infrared fluorescent dye, and physiological saline as a dispersion medium. In addition to these components, for example, a pH adjusting agent, An organic solvent such as ethyl alcohol, a viscosity modifier, a surfactant, a stabilizer, an antioxidant, a chelating agent, a colorant, and the like may be contained in a trace amount or an appropriate amount.

  Moreover, although it is a composition ratio, when the weight of the phospholipid which comprises a liposome is set to 1, the near-infrared fluorescent pigment | dye is 0.001-0.1 especially 0.002-0.005 more preferable. When the liposome particles are micrometer size, the phospholipid is preferably a mixture of saturated fatty acid type phospholipid and unsaturated fatty acid type phospholipid. In that case, saturated fatty acid type phospholipid: unsaturated fatty acid type phospholipid The lipid ratio is preferably 1: 5 to 5: 1 by weight, and most preferably 1: 1.

  Further, the amount of gas encapsulated in the liposome particles is preferably 0.1 to 10, particularly preferably 0.5 to 1, for example, assuming that the weight of the phospholipid constituting the liposome is 1.

  In the present invention, the particle size (diameter) of the liposome particles is essential to be nanometer or micrometer size. In the former case, it is preferably several tens to several hundreds of nm, particularly 50 to 200 nm. 8-20 micrometers is preferable especially 1-5 micrometers.

  The use of the contrast agent of the present invention varies depending on the particle size. In the case of nano size, it is used for imaging (contrast, imaging, observation) of lymphatic system (lymph vessel, lymph node). Used for imaging (contrast, imaging, observation).

  The physiological saline as a dispersion medium is an aqueous solution (saline) of sodium chloride that is almost isotonic with body fluid. For physiological saline, a pH adjuster, an organic solvent such as ethyl alcohol, and a viscosity adjuster are appropriately used. , A surfactant, a stabilizer, an antioxidant, a chelating agent, a coloring agent, a glucose aqueous solution, and the like may be contained in a trace amount or in an appropriate amount.

  In addition to gas encapsulated in liposome particles, drugs such as anticancer drugs, anti-inflammatory drugs, corticosteroids, antiviral drugs, antibiotics, antituberculosis drugs, various molecular targeted therapeutic drugs (anti-VEGF antibody, anti-EGFR2 antibody), antithrombosis You may include medicine.

  By encapsulating the drug, the contrast medium of the present invention can also be used for DDS. After the near-infrared fluorescence contrast method is used to guide to the correct position, and it is recognized that the lesion part (local area) in the deep part has been reached by the ultrasonic contrast method, an external stimulus such as strong ultrasound is irradiated. The drug can be released by destroying the liposome particles.

  In the method for producing the dual-purpose contrast agent of the present invention, a near-infrared fluorescent dye may be added before preparing the liposome-based ultrasonic contrast agent (raw material preparation stage) or after the preparation. Liposome production apparatuses are also commercially available, and production is not difficult.

  As shown in FIGS. 1 and 7, the dual-use contrast medium of the present invention is a dispersion (suspension) in which liposomes are dispersed in physiological saline as a dispersion medium. It is a feature of the present invention that a dye is bound to the liposome. This bond may not be a chemical bond but may be an ionic bond, a hydrogen bond, a hydrophobic bond, a protein-substrate bond, a protein-sugar chain bond, or the like.

Example 1
(1) Preparation of dual-use contrast agent (abbreviated as “fluorescent nanobubble”) First, weigh 22.85 mg of phosphatidylcholine (DSPC), 3.48 mg of DSPE-PEG2000, 0.70 mg of ICG-C18, and screw them Put in. Both are in powder form. DSPE-PEG2000 is “1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [cyanur (polyethylene glycol) -2000]”.

  Next, for dissolution, chloroform and methanol are mixed at a volume ratio of 4: 1 to prepare a mixed solvent, and 5 ml of the mixed solvent is added to the screw tube to dissolve the powder therein. did.

  Set the screw tube containing this solution in a vortex mixer GENIE-2 product number G560 (product of US Scientific Industries), stir (strength 10), and when completely dissolved, screw the screw tube After removing the lid of the formula, the opening of the screw tube was covered with aluminum foil. Then, the volatilization port is opened with a needle in the aluminum foil, the screw tube is put into a poly desiccator (300 type, product of ASONE CORPORATION), and the vacuum pump (N810FT.18, product of KNF Japan Co., Ltd.) is installed. The organic solvent was volatilized for 300 minutes.

After confirming that the organic solvent has completely volatilized and a thin film has formed on the bottom of the screw tube, remove the screw tube from the polydesiccator, and then prevent the PBS solution (phosphate buffered saline) from peeling off the thin film. Carefully, 5 ml of PBS solution was added, and left in a thermostat set at 90 ° C. for 5 hours.
After the standing was completed, the thin film was swollen by gently vibrating the screw tube and stirring the inside.

  The suspension thus obtained was passed through a 400 nm membrane filter using a gas tight syringe holder set for particle size adjustment (mini-extruder set for liposome production, a product of Avanti Polar Lipids, Inc., USA). The particle size was shaped.

  After filling the particle shaped solution into 4 ml of a “10 ml vial”, plugged with a rubber cap, an aluminum cap was put on and completely closed.

  Then, 15 ml of air as gas was pressurized into the vial using a luer lock syringe (syringe).

  As soon as pressurization was completed, the vial was placed in an ultrasonic bath filled with degassed water and irradiated with ultrasonic waves for 5 minutes.

  Thus, a dual-use contrast agent (abbreviated as “fluorescent nanobubble”) having an average liposome particle diameter (diameter) of 49.4 nm was obtained.

  An image of this “fluorescent nanobubble” is shown in FIG. In FIG. 1, reference numeral 1 represents liposome particles, reference numeral 2 represents gas encapsulated in the liposome particles, reference numeral 3 represents a pigment, and reference numeral 4 represents a PBS solution (phosphate buffered saline).

  FIG. 2 shows the volume distribution of liposome particles of this “fluorescent nanobubble” measured by a particle size measuring device (product number Nanotrac UPA-UT151, a product of Microtrac Bell Co., Ltd.).

(2) Comparative Example 1
Comparative Example 1 is nanobubbles (suspension) to which ICG-C18 is not bonded. Except for the point that ICG-C18 is not bonded, the preparation method is the same as that of the dual-use contrast agent (abbreviated as “fluorescent nanobubble”) of Example 1. The average particle diameter (diameter) of the liposome particles is 205.2 nm, and the gas encapsulated in the liposome particles is air.

(3) Evaluation
(A) Phantom experiment A simple flow path system was constructed by combining a small water tank as shown in Fig. 3 (side view). Although only one channel is visible in FIG. 3, there is another channel in the depth direction. The flow path is a hollow silicon tube (diameter φ = 2 mm). The fluorescent nanobubbles of Example 1 and the nanobubbles (suspension) of Comparative Example 1 are circulated in the respective channels, and a near-infrared fluorescence observation apparatus (part number HyperEye Medical Systems) denoted as “near-infrared fluorescent probe” in FIG. As with II, a product of Mizuho Medical Co., Ltd.), each was photographed using an ultrasonic diagnostic imaging device (Part No. Aplio500, a product of Toshiba Medical Systems Co., Ltd.) labeled “Ultrasonic probe”. The result is shown in FIG.

  FIG. 4 shows a fluorescence image of a flow path observed and photographed from above using the near-infrared fluorescence observation apparatus when Example 1 and Comparative Example 1 are circulated [FIGS. 4 (1), (2), ( 5), (6)] and a tomographic image of the flow path photographed by the ultrasonic diagnostic apparatus [FIGS. 4 (3), (4), (7), (8)].

  4 (1), (3), (5), and (7) were taken immediately after the start of circulation, and (2), (4), (6), and (8) were taken 15 or 19 hours after the start of circulation. It is an image. From the comparison of (1) and (5), in Comparative Example 1 where ICG-C18 is not bonded, the flow path is not fluorescent, in Example 1, the flow path is fluorescent, and Example 1 is near-infrared fluorescence observation. It can be seen that it functions as a contrast medium. Moreover, from the comparison between (3) and (7), it can be confirmed that the inside of the flow path where the ultrasonic contrast agent is present is observed (contrast) in both Comparative Example 1 and Example 1. It can be seen that the function of the ultrasonic contrast agent of Example 1 to which ICG-C18 is bonded is comparable to that of Comparative Example 1 to which ICG-C18 is not bonded. From the comparison of (5) and (6) and (7) and (8), it can be seen that Example 1 retains the function as a dual-purpose contrast agent even after 10 hours or more after circulation.

(B) Animal Experiment The fluorescent nanobubbles of Example 1 were subcutaneously injected in vivo into the pig foot for animal experiment shown in (1) of FIG. As a result, the fluorescent nanobubbles naturally flow into the lymph nodes via the lymphatic vessels.

  Then, since it image | photographed using the said near-infrared fluorescence observation apparatus, the obtained fluorescence image is shown to (2) of FIG. In this fluorescent image (2), a portion that appears strongly white and triangular is an injection site, and a high-luminance site described linearly from the site is a lymph vessel.

  Further, since a site where a lymph node is present was photographed, this appearance photograph is shown in (3) of FIG. 5, and a near-infrared fluorescence image is shown in (4). It can be confirmed that a plurality of linear lymphatic vessels flow into the lymph nodes. Thus, the spatial distribution of fluorescent nanobubbles of Example 1 (that is, the distribution of lymphatic vessels) can be clearly observed as a fluorescent site, and there is no problem in the function as a contrast agent for near-infrared fluorescence observation even in living tissue. It was confirmed.

  The moment when the fluorescent nanobubbles of Example 1 flow into the lymph nodes from the body surface of the site where the lymph nodes are present through the lymph vessels is observed with the above-described ultrasonic diagnostic imaging apparatus, and an ultrasonic image shown in FIG. Got. In addition, since the same imaging | photography was performed previously before injecting the fluorescent nanobubble of Example 1 subcutaneously, the imaging | photography image is shown in FIG. 6 (1). It was confirmed that the pharmacokinetics of Example 1 can be confirmed in real time at low echo intensity sites such as lymph nodes.

  As described above, the following can be understood from the fluorescence image of the near-infrared fluorescence contrast (FIG. 5) and the image of the ultrasound contrast contrast (FIG. 6). With near-infrared fluorescence contrast, it is possible to know the travel of a lymph vessel over a wide range (macro travel), and with ultrasound contrast, it is possible to know the local (micro) internal structure of a lymph node. That is, the fluorescent nanobubble of the present invention enables multi-scale observation with a single contrast agent injection for the same site.

Example 2
(1) Preparation of dual-use contrast agent (abbreviation “fluorescence microbubble”) 15 mg of phosphatidylcholine (DSPC), 7.6 mg of egg yolk lecithin, and 0.70 mg of ICG-C18 were weighed and transferred to a mill container.

  The mill container was immersed in liquid nitrogen and frozen. This mill container was then set in a mixer mill and the frozen material in the container was pulverized. The grinding condition is “20 frequency / sec” for 2 minutes.

  Then, the above freezing and pulverization were repeated once again.

  The pulverized product was transferred to a beaker, 5 ml of PBS solution was poured into the beaker, and stirred and mixed using a magnetic stirrer (400 rpm) to prepare a solution (phospholipid solution).

  To this phospholipid solution, 5 ml of PBS solution and 10 mg of DSPE-PEG2000 were added and stirred for 6 hours with a magnetic stirrer (400 rpm) to prepare a phospholipid mixed dispersion (dispersion containing liposome particles).

  Next, the dispersion liquid is sucked up with a syringe (syringe), and further, air is put into the syringe at a “dispersion liquid: air volume ratio of 2: 1”, and the syringe is shaken vigorously by hand to remove the dispersion liquid and air. Stirring was performed, thereby producing a bubble dispersion.

Finally, the bubble dispersion is stirred with an ultrasonic homogenizer to shape the particle size. Thus, the liposome average particle size (diameter): 2.4 μm (= radius 1.2 μm × 2), liposome particle concentration: 5.53 × 10 ⁴ particles / mm ³ of a dual-purpose contrast medium (abbreviation “fluorescent microbubble”) was obtained.

  An image of this “fluorescence microbubble” is shown in FIG.

(2) Comparative Example 2
Comparative Example 2 is a liposome particle (suspension) to which ICG-C18 is not bound. Except for the point that ICG-C18 is not bonded, the preparation method is the same as that of the dual-purpose contrast agent (abbreviated as “fluorescence microbubble”) of Example 2. The main component of this contrast agent is a liposome suspension, the dispersoid is liposome particles, and the dispersion medium is a PBS solution. The average particle diameter (diameter) of the liposome particles is 2.2 μm (radius 1.1 μm × 2), and the liposome concentration is 6.65 × 10 ⁴ particles / mm ³ . The gas encapsulated in the liposome particles is air.

  FIG. 11 shows an optical microscope in which (1) the contrast agent of Example 2 and (2) the liposome particles (suspension) of Comparative Example 2 are each connected with a 100 × objective lens (product number UPlansApo, product of Olympus Corporation). It is a bright-field image (photograph) observed using (Product No. BX51, Olympus product). It can be seen that the liposome particles of Example 2 and Comparative Example 2 are spherical.

  The above-mentioned optical microscope has an epifluorescence system, a xenon light source (part number U-LH75XEAPO, a product of Olympus Corporation), and an ultra-high sensitivity EMCCD camera to enable near-infrared fluorescence observation in addition to normal bright-field observation. (Product number iXons3, product of ANDOR TECHNOLOGY) is added, and bright field observation (visible light observation mode) and fluorescence observation (fluorescence observation mode) can be switched on the software. That is, a bright field and near-infrared fluorescence microscopic image of the same object can be acquired.

  FIG. 12 is a fluorescence image (photograph) acquired in the fluorescence observation mode. (1) The photograph of Example 2 is bright in the shape of a sphere, whereas (2) the photograph of Comparative Example 2 is black and nothing is shown. In the photograph of FIG. 12 (2), the image is taken under the same observation (fluorescence imaging) conditions (setting of excitation light intensity, gain, etc.), but the fluorescence image is not confirmed.

From comparison between (1) and (2) in FIG. 12, it can be confirmed that ICG-C18 is bound to the phospholipid membrane of the liposome particles. In addition, when (1) <two-dimensional photograph> of FIG. 12 is observed closely, it shines brightly in an annular shape, but does not shine inside the annular zone. This is because the depth of field of the microscope system, that is, the observation range in the depth direction with respect to the two-dimensional photograph is limited, and the fluorescence of ICG-C18 on the surface portion of the phospholipid film inside the annular zone is not observed.
The position of the liposome particles slightly differs between the bright field observation image (FIG. 11) and the near-infrared fluorescence image (FIG. 12), because the liposome particles moved during observation.

(3) Measurement of average particle size, particle concentration and particle size distribution For “fluorescent microbubbles” in Example 2 and Comparative Example 2 (liposome suspension), the average particle size, particle concentration (number density) of particles and The particle size distribution was measured. 20 μl of each was prepared, injected into a hemocytometer (Thoma board, product number 2-5552-31, product of ASONE CORPORATION), and observed with an optical microscope. The average particle size, particle concentration (number density), and particle size distribution were determined by measuring the number and radius of particles in a predetermined volume and then calculating. The result is shown in FIG.
The particle concentration (number density), average particle diameter (radius) ± standard deviation of “fluorescent microbubble” of Example 2 is 5.53 × 10 ⁴ / mm ³ , 1.2 ± 0.49 μm, and is a comparative example. 2 is 6.65 × 10 ⁴ / mm ³ , 1.1 ± 0.53 μm, which is almost equal.

(4) Evaluation
(A) Phantom experiment The simple flow path system (see FIG. 3) used in Example 1 was used. The “microbubble” of Example 2 and Comparative Example 2 were circulated through two channels (diameter φ = 2 mm), respectively, and a near-infrared fluorescence observation apparatus (product number HyperEye Medical Systems II, a product of Mizuho Medical Co., Ltd.) And an ultrasonic diagnostic imaging apparatus (Aplio500, a product of Toshiba Medical Systems), respectively. The result is shown in FIG.

  FIG. 8 shows Example 2, Comparative Example 2, and PBS (physiological saline). The former two were circulated in the channel in the first experiment and the latter PBS was circulated in the second experiment. Fluorescence image [(2), (3)] of the channel when observed and photographed from above the simple channel system (FIG. 3 = side view) using the apparatus and the channel when photographed by the ultrasonic diagnostic imaging apparatus The tomographic images [(5), (6)]. In addition, from FIGS. 8 (2) to (3), Comparative Example 2 and PBS (physiological saline) that do not contain or contain ICG-C18 do not show any channel (fluoresce) at all. On the other hand, in Example 2, the flow path is reflected (fluoresced), and it can be seen that Example 2 functions as a contrast agent for near-infrared fluorescence observation.

  Further, from FIG. 8 (6), it can be seen that in Example 2 and Comparative Example 2, the flow path in which the contrast agent is present is observed and contrasted as a high-luminance part of ultrasonic contrast. On the other hand, as shown in (5), it can be seen that in PBS (physiological saline), the flow path (more precisely, the inside of the flow path) is observed and contrasted as a low-luminance part of ultrasonic contrast. However, the wall of the channel is observed as a high luminance part.

  As a result, it can be seen that Example 2 to which ICG-C18 is bonded has the same function as an ultrasonic contrast agent compared to Comparative Example 2 to which ICG-C18 is not bonded.

(B) Animal experiment (a) A chicken breast for meat is prepared, and as shown in FIG. 9, the contrast medium of Example 2 is injected with a syringe at a depth of 1 mm, 5 mm, and 15 mm. Ultrasonographic imaging and near-infrared fluorescence imaging were performed. The former used a near-infrared fluorescence observation apparatus (product number HyperEye Medical Systems II, a product of Mizuho Medical Co., Ltd.), and the latter used an ultrasound diagnostic imaging apparatus (product number Aplio500, a product of Toshiba Medical Systems).

  The result is shown in FIG. FIG. 10 (1) is an appearance photograph of the chicken breast observed and photographed from outside the chicken breast with a digital camera after contrast medium administration, and FIG. 10 (2) is the outside of the chicken breast with the near infrared fluorescence observation apparatus. It is the fluorescence image image | photographed and acquired from. Although it can be seen that the administration site was brightly fluorescent at the site where Example 2 was administered at a depth of 1 mm and 5 mm from the surface, no clear fluorescence was observed at the site administered at a depth of 15 mm. It was confirmed that Example 2 functions as a contrast agent for near infrared fluorescence observation at a site shallower than 10 mm from the surface of the living tissue. This is about the same performance as normal ICG.

  FIG. 10 (3) is a tomographic image of a chicken breast observed with an ultrasonic diagnostic apparatus (Part No. Aplio500, product of Toshiba Medical Systems) before contrast medium administration. The tip of the syringe can be visually recognized as a bright linear structure at a position 15 to 17 mm deep from the surface. The bright part existing at a depth of 17 to 20 mm from the surface is the boundary between the chicken breast and the base. FIG. 10 (4) is a tomographic image obtained by observing the same part using the same device after administration of the contrast medium. It turned out that the brightness increased centering on the syringe tip confirmed before contrast agent administration, and it confirmed that the contrast agent "fluorescence microbubble" of a present Example functions as an ultrasonic contrast agent.

  It is also possible to inject the contrast medium of Example 2 directly into the artery or portal vein of the pig liver under laparotomy and observe the internal vasculature from the liver surface.

  As described above, the dual-purpose contrast medium of the present invention is itself deeply injected when it is injected into a living tissue. Therefore, the near-infrared fluorescence imaging has a living tissue depth of about 10 mm, and the ultrasonic imaging has a living body. Imaging (observation) is possible up to a tissue depth of about 30 mm.

  Therefore, it is possible to observe both up to about 10 mm in depth by both near-infrared fluorescence contrast imaging and ultrasonic contrast imaging, and it is possible to observe only from about 10 mm to 30 mm depth by ultrasonic contrast imaging. In a region that can be observed in duplicate (up to a depth of about 10 mm), specify the location (affected area) that you want to see with near-infrared fluorescence contrast, which is good for wide-range observation, and this location (affected area) It is possible to do convenient operations such as viewing with sonography. In this case, the conventional near-infrared fluorescent contrast agent has a small molecule, so it is difficult to diffuse in the tissue and the residence time is short. However, the dual-purpose contrast agent of the present invention has a near-infrared fluorescent contrast agent portion in the liposome particle. Since it is bonded, it is large, and therefore has the advantage of a long residence time. The residence time is longer for microbubbles than for nanobubbles.

  The contrast agent capable of both ultrasonic imaging and near-infrared fluorescence imaging of the present invention is a deep part (specifically, liver, kidney, pancreas, gallbladder, stomach, stomach, duodenum, skin, etc.) Since it reaches microvascular systems and lymphatic systems having a depth of 0 to several tens of millimeters), it is possible to obtain images by macroscopic and microscopic multiscale imaging of their distribution. Therefore, it can be used for pathological diagnosis and research of living tissue (affected area). Therefore, it may be used in the reagent industry, pharmaceutical industry, hospital, university, laboratory, etc.

1 .... liposome particle 2 .... gas 3 .... dye 4 .... physiological saline (PBS)

Claims (1)

An ultrasonic contrast agent (A) comprising a liposome particle as a dispersoid and physiological saline as a dispersion medium, the diameter of the liposome particle being nano-sized or micro-sized, and containing a gas inside the liposome particle, and near red It consists of a near-infrared fluorescent contrast agent (B) consisting of an outer fluorescent dye,
A contrast agent capable of both ultrasonic contrast and near-infrared fluorescence contrast, wherein the liposome particles and the near-infrared fluorescent dye are bound to each other.