JP3896176B2 - Near-infrared fluorescent tracer and fluorescent imaging method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a near infrared fluorescent tracer.
[0002]
[Prior art]
A technique for measuring the internal state of a thick biological sample (human body, etc.) using light is important for medical research and diagnosis / treatment of diseases. In particular, knowing the position and size of a tumor in advance by image diagnosis is an important technique for removing a tumor, and several methods are already known.
[0003]
For example, there is a problem that image diagnosis of a tumor tissue or a nerve axillary flow using a radioisotope (RI) has a problem of exposure and contamination and is complicated to manage. Moreover, since it cannot be used outside the management area, there is a problem that it is difficult to apply it during surgery. For example, when a substance is administered in the vicinity of the neuromuscular junction, a phenomenon is known in which nerve cells take up the substance and actively transport it from the nerve terminal toward the cell body (nerve axon flow). It is known that when the flow speed is lower than normal, the nerve cell can be diagnosed as having some kind of disorder. In fact, attempts have been made to use RI for this diagnosis, but it has the same problems as tumor diagnosis.
[0004]
The X-ray CT technology has the same exposure problems as those of radioisotopes, and the device is large and the subject must be placed inside the tomography device. There is also a problem that is difficult.
[0005]
[Problems to be solved by the invention]
The present invention has been made in view of the above problems. That is, the present invention provides a new tracer that can be used for a low-toxicity living body for measuring the internal state of a thick biological sample (human body, etc.) in vitro by using near-infrared fluorescence. A measurement imaging method is provided.
[0006]
[Means for Solving the Problems]
Since near-infrared light is highly permeable to living organisms, it is considered that pigments that emit near-infrared fluorescence are distributed in the body and measured outside the body, and can be applied to various diagnoses and the like. Further, this method can be realized only with a small and inexpensive apparatus such as a halogen lamp, a CCD camera, an optical filter and a lens.
[0007]
On the other hand, when used as a tracer in a living body, its toxicity becomes a problem, and there is a limit to the dye that can be used. Because of this toxicity problem, indocyanine green has actually been clinically applied as one of the tracer dyes that can be used. This dye emits near-infrared fluorescence (835 nm) in an organic solvent, and there is an example of using this to observe fluorescence of the fundus blood vessel.
[0008]
However, this dye is fluorescent in a nonpolar solvent (such as an organic solvent), but is non-fluorescent in a polar solvent (such as an aqueous solution), and cannot be used as it is.
[0009]
The present inventor conducted intensive research in view of the above points and succeeded in finding a novel near-infrared fluorescent tracer having a non-toxic near-infrared fluorescent dye and water-soluble. In other words, it was found that a dye having low toxicity such as indocyanine green but not substantially fluorescent in an aqueous solution becomes fluorescent by forming a complex with an appropriate high-density lipoprotein, etc. Based on this, the complex was successfully used as a near-infrared fluorescent tracer. In addition, a near-infrared fluorescent dye tracer that further has a recognition unit that can specifically recognize an object to be detected is found, and based on this knowledge, the complex is a near-infrared fluorescent tracer that specifically recognizes the object to be detected. succeeded in.
[0010]
More specifically, the present invention provides a near-infrared fluorescent tracer comprising a complex having at least a near-infrared fluorescent dye and an R substance containing a fat-soluble component.
[0011]
The present invention also provides a near-infrared fluorescent tracer comprising at least a near-infrared fluorescent dye, a substance containing a fat-soluble component, and an object recognition unit.
[0012]
Furthermore, the present invention provides a near-infrared fluorescent tracer characterized in that the infrared fluorescent dye is an indocyanine green dye, and the substance containing the fat-soluble component is a high-density lipoprotein.
[0013]
The present invention also provides a near-infrared fluorescent tracer characterized in that the object recognition portion is an antibody.
[0014]
Furthermore, the present invention provides a method for in vitro fluorescence imaging by introducing a near-infrared fluorescent tracer into a living body (excluding humans) , irradiating the living body with excitation light, and detecting near-infrared fluorescence from the tracer. Is.
[0015]
Hereinafter, the present invention will be described in more detail with reference to embodiments.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
(Near-infrared tracer)
The near-infrared tracer according to the present invention contains a dye having a fluorescence wavelength in the near-infrared region, and can be traced by detecting the fluorescence. Here, the preferred near infrared region is 700 nm, more preferably 800 nm or more, and the upper limit is not particularly limited, but in an actual organic dye, it is in the range of about 1200 nm to 1600 nm. The dye itself needs to be fluorescent at least in a medium in which an object to be detected exists, but does not necessarily have to be fluorescent in an aqueous solution as described below.
[0017]
In addition, when the tracer according to the present invention is used in a living body, it is desirable that it is water-soluble (dissolvable in an in-vivo liquid medium) and has no toxicity to the living body. In the present invention, there is no particular limitation as long as the dye has both of the above two characteristics, but in the present invention, for example, an indocyanine green dye can be preferably used. In fact, indocyanine green pigments are water-soluble and are used as drugs for examining hepatic circulation function, and there is no problem in terms of toxicity.
[0018]
Furthermore, the near-infrared tracer according to the present invention is preferably a complex in which the dye is bound to another biological component. Other biological components to which the dye can bind are not particularly limited, but various biological components can be selected depending on the purpose of use of the tracer according to the present invention. For example, (1) when the dye is insoluble in an in vivo liquid medium (for example, blood, spinal fluid, etc.), it is possible to form a complex with various biological components for solubilization, 2) Conversely, although the above dye is soluble in an in vivo liquid medium (for example, blood, spinal fluid, etc.), it is not sufficiently fluorescent in a water-soluble in vivo liquid medium, rather it is a non-aqueous solvent. When it becomes fluorescent, it can be made fluorescent by forming a complex with various biological components. (3) Further, as described below, the tracer according to the present invention is covered. It is also possible to form a complex with an appropriate biological component in order to introduce a part that specifically recognizes the detected substance.
[0019]
In fact, the above indocyanine dyes are not problematic at all in terms of toxicity, but are substantially non-fluorescent in water-soluble solvents. Therefore, as shown in the examples of the present invention, the indocyanine dye is formed into a complex with a high-density lipoprotein, which is a biological component having a fat-soluble component, so that the indocyanine dye is contained and the indocyanine dye is fluorescent. Can be sex.
[0020]
Furthermore, the near-infrared tracer according to the present invention preferably has a part that specifically recognizes the detection object in addition to the above-mentioned dye complexed with another biological component. The recognition unit only needs to specifically recognize an object to be detected, and is not particularly limited. For example, the well-known one based on protein-protein interaction, such as one based on antigen-antibody binding, receptor ligand binding, or the like is possible. Furthermore, the detection object is not particularly limited, but various in vivo cells (for example, cancer cells) are possible. The method for providing the recognition unit is not particularly limited, but, for example, a bond formation reaction used in an ordinary protein chemical modification reaction can be preferably used. In the case described above, a tracer having a recognition unit that includes a fluorescent dye and specifically recognizes an object to be detected is configured.
[0021]
(Imaging using near infrared tracer)
The near-infrared tracer according to the present invention is soluble in an in-vivo liquid medium in vivo, and has fluorescence in the near-infrared region. Therefore, the tracer is introduced into the living body, and the tracer moves in the living body by diffusion or fluid flow of the body fluid, and its position and concentration change are imaged in real time by observing the fluorescence based on the tracer from outside the living body. It becomes possible to do.
[0022]
For example, by chemically binding an anti-tumor antibody to an indocyanine green-high density lipoprotein (ICG-HDL) complex, the position and size of an external tumor in vivo can be measured in vitro in real time. . At this time, since the imaging apparatus is small and can be measured in real time, it can also be used during the resection of the tumor. At this time, since the tumor tissue and the normal tissue can be discriminated on the spot, the normal tissue is not cut wastefully or the tumor tissue is not left uncut.
[0023]
In addition, by labeling antibodies against other surface antigens (receptor proteins and viral coat proteins that appear on the surface of tissues), it is possible to detect which part of an organism, not just a tumor, is present in an organism, and to be infected with a virus. Thus, any tissue can be easily and specifically recognized, and can be used for diagnostic reagents and diagnostic methods for various diseases.
[0024]
For example, as an image diagnosis of nerve axon flow, if the ICG-HDL alone is administered in the vicinity of the neuromuscular junction, it is taken up by nerve cells, rides on the axon flow, and measures the fluorescence obtained in vitro. Flow measurements are observable.
[0025]
There is no particular limitation on an apparatus for measuring the fluorescence from the tracer in vitro using the tracer according to the present invention. However, an optimal excitation light can be obtained by using a normal excitation light source and, if necessary, a filter for the excitation light source. The fluorescence from the dye produced by the excitation light is detected by a fluorescence detector. If necessary, a filter can be used to select only the fluorescence from the dye. Further, it is preferable to image the obtained fluorescence information by data processing. In this case, the data processing apparatus is not particularly limited. For example, “ARGUS20 (manufactured by Hamamatsu Photonics)” can be used.
[0026]
Furthermore, the position and size of the tumor can be measured in vitro by chemically binding the antitumor antibody to the tracer according to the present invention, for example, ICG-HDL, by the imaging described above. If this technique is performed, for example, during surgery, it can be used to develop an apparatus for preventing the normal tissue from being cut unnecessarily or leaving the tumor tissue excised by accurately distinguishing the tumor tissue from the normal tissue. . At this time, in order to bind the antibody to ICG-HDL, as already described, for example, a method of binding an HDL moiety and an antibody (crosslinking reaction between proteins, usually crosslinking between amino groups with glutaraldehyde, etc.), or A method of cross-linking the ICG moiety to the amino group of the antibody is possible.
[0027]
(Example)
As described below, a complex (ICG-HDL) of a near-infrared fluorescent dye indocyanine green (ICG) and human high-density lipoprotein (HDL) was used. HDL used here is a protein component in blood in which lipid and protein are combined. ICG dissolves in the lipid portion of the HDL as described above, resulting in fluorescence. The resulting complex is soluble in water, so ICG-HDL can be administered to various parts of the living body, and its near-infrared fluorescence is observed from outside the body.
[0028]
Since ICG is actually administered to the human body in clinical examinations, and HDL is also a biological component from the beginning, even if this complex is administered to the human body from the outside, there is little toxicity.
[0029]
(1) 20.5 mg of ICG (Diagono Green, Daiichi Pharmaceutical) was dissolved in 4 ml of distilled water to prepare an aqueous solution of about 5.1 mg / ml (= 5.54 mM).
[0030]
To 250 μl of human high density lipoprotein solution (HDL, manufactured by Kappel, 20 mg / ml), 2.5 μl of ICG aqueous solution was added and stirred to prepare a dye-protein complex (ICG-HDL). The ICG concentration in this complex solution is about 55.4 μM.
[0031]
(2) Optical characteristics of ICG-HDL.
[0032]
The above ICG-HDL was diluted 50 times with phosphate buffered saline (PBS), and the fluorescence spectrum (uncorrected) was measured. For comparison, the spectrum of an organic solvent (DMSO) solution having the same concentration was also measured. The results are shown in FIG. 1 (in addition, it is an uncorrected spectrum, and the fluorescence maximum value is a literature value (A. Schneider, A. Kaboth, L. Neuhauser: Detection of subretinal neovascular membranes with indocyanine green and an infrared scanning laser ophthalmoscope, American J. of Ophthalmol., 113-1, 45/51 (1992)).
[0033]
From this result, it is clear that the ICG-HDL complex shows the same fluorescence spectrum as that of the DMSO solution. That is, ICG-HDL exhibits a fluorescence intensity of 58.3% (wavelength 835 nm) with respect to a DMSO solution.
[0034]
(3) Near infrared fluorescence imaging using ICG-HDL.
[0035]
The obtained ICG-HDL was administered to a live animal, and the near-infrared fluorescence of ICG was imaged, and ICG-HDL was distributed in the body in vitro. FIG. 2 shows an outline of the measurement system used in this example. That is, after introducing the ICG-HDL tracer 4 into the experimental animal 3, a 150 W halogen lamp is attached to the Bunt Bass filter (center wavelength 720 nm) to form the excitation light source 1, and this light is transmitted to the experimental animal 3 via the optical fiber 2. Irradiate. A CCD camera (C2400-75i manufactured by Hamamatsu Photonics) 6 equipped with a TV lens (FUJINON CF8A1: 1.8 / 8) was used to detect the near-infrared fluorescence of the generated ICG-HDL. However, the infrared cut filter of the CCD camera is removed, and a sharp cut filter (transmitted wavelength 840 nm or more) 5 is set on the TV lens. The signal from the CCD camera 6 is captured by an image processing device 7 (ARGUS20 (manufactured by Hamamatsu Photonics)).
[0036]
That is, 25 μl of ICG-HDL solution was injected into the right brain of a 3-day-old Wistar rat (male) with a syringe. FIG. 3 shows a reflected light image (without a sharp cut filter) and a near-infrared fluorescent image (with a sharp cut filter).
[0037]
FIG. 3 is an image of the head immediately after administration (the exposure time of the CCD camera in the fluorescence image is 1 second). It can be seen that ICG-HDL is distributed in the frontal region around the administration site. FIG. 4 is an image of the head 1 hour after administration (exposure 1 second). It can be seen from the fluorescence image that the distribution of ICG-HDL has moved to the back of the head. FIG. 5 is an image of the whole body 8 hours after administration (right is head, left is tail, exposure 8 seconds). It can be seen that ICG-HDL has moved to the end of the spinal cord. Further, FIG. 6 schematically shows the result of the above time change in order to clarify the relationship between the obtained fluorescence intensity and time.
[0038]
This result clearly shows that ICG-HDL can be observed over time in living experimental animals as it moves deeper than the body surface from the brain into the spinal cord. This also indicates that ICG-HDL can be used as a tracer for observing a biological sample having a thickness.
[0039]
【The invention's effect】
The structure of the tracer according to the present invention, that is, a complex of a near-infrared fluorescent dye and an appropriate biological substance is soluble in body fluids and cannot be used as a fluorescent dye in an aqueous solution. A certain less toxic dye is fluorescent even in an aqueous solution. Therefore, by using the tracer according to the present invention together with the fluorescence detection device (excitation light source, detection system such as a CCD camera, and image processing device), it is possible to measure and image in vitro.
[0040]
That is, an X-ray source, a laser light source, a tomography detector, a high-speed computer, etc. are not necessary, and equivalent imaging is possible. Furthermore, since it is simple and inexpensive, real-time imaging (for example, during surgery) can be applied.
[0041]
In addition, in the angiography using ICG alone, the adaptation site is limited, but there is no such limitation if ICG-HDL is used.
[Brief description of the drawings]
FIG. 1 is a diagram showing a fluorescence spectrum of an indocyanine green dye in DMSO and a fluorescence spectrum in an aqueous solution of an indocyanine-high density lipoprotein complex according to the present invention.
FIG. 2 is a diagram showing an outline of a measurement system for imaging using an indocyanine-high density lipoprotein complex according to the present invention as a tracer.
FIG. 3 is a photomicrograph showing a reflected light image (upper) and a near-infrared fluorescent image (lower) immediately after administration of a rat administered with ICG-HDL.
FIG. 4 is a photomicrograph showing a reflected light image (upper) and a near-infrared fluorescent image (lower) after 1 hour of administration of a rat administered with ICG-HDL.
FIG. 5 is a photomicrograph showing a reflected light image (top) and a near-infrared fluorescent image (bottom) 8 hours after administration of a rat administered with ICG-HDL.
FIG. 6 is a diagram schematically showing the relationship between the obtained fluorescence intensity and time, as a result of time change after administration of a rat administered with ICG-HDL.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Excitation light source, 2 ... Optical fiber, 3 ... Sample (rat newborn), 4 ... ICG-HDL tracer, 5 ... Sharp cut filter (840 nm), 6 ... Detector (camera), 7 ... Image processing apparatus, 8 ... Fluorescence

Claims (7)

  A near-infrared fluorescent tracer comprising a complex having at least a near-infrared fluorescent dye and a substance containing a fat-soluble component. The near-infrared fluorescent tracer according to claim 1, wherein the near-infrared fluorescent dye is an indocyanine green dye, and the substance containing the fat-soluble component is a high-density lipoprotein. An in vitro fluorescence imaging method by introducing the near-infrared fluorescent tracer according to claim 1 or 2 into a living body (excluding a human), irradiating the living body with excitation light, and detecting near-infrared fluorescence from the tracer. A near-infrared fluorescent tracer comprising at least a near-infrared fluorescent dye, a substance containing a fat-soluble component, and an object recognition unit. The near-infrared fluorescent tracer according to claim 4, wherein the near-infrared fluorescent dye is an indocyanine green dye, and the substance containing the fat-soluble component is a high-density lipoprotein. 6. The near-infrared fluorescent tracer according to claim 4 or 5, wherein the object recognition unit is an antibody. By introducing the near-infrared fluorescent tracer according to any one of claims 4 to 6 into a living body (excluding a human), irradiating the living body with excitation light, and detecting near-infrared fluorescence from the tracer. In vitro fluorescence imaging method.

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