Patent Application
20150328343
The present invention relates to a contrast medium, and a method and kit for preparing the contrast medium.
Diagnostic imaging modality such as X-ray CT, MRI and ultrasonic diagnostic equipment has long been used as an essential tool at medical front. In these tools, imaging is made based on difference in in-vivo CT values, spin relaxation time and acoustic impedance. Since difference in these physical properties exclusively reflects a structure (figure) of a living body, the modality is called “form imaging”.
In contrast, imaging of a site of the same structural tissue but different in function is called “functional imaging”. Particularly in the functional imaging, visualizing the present states of molecules, such as proteins, constituting a living organism, is often called “molecular imaging”. The molecular imaging is expected to be used in understanding life phenomena such as development and differentiation and applied to diagnosing and treating diseases. Because of this, the molecular imaging is a research field currently most receiving attention.
In the molecular imaging, a “molecular probe” is frequently used, which is a substance having structural selectivity to molecules constituting a living organism. In this case, to the molecular probe, a detectable structure by any physical means is added and the distribution of the molecular probe in a body is visualized. Attempt to use such molecular imaging in early diagnosis has been accelerated in recent years.
In an attempt to use modality such as MRI and ultrasonic diagnostic equipment, which has been previously used in form-imaging, in the molecular imaging, research and development are conducted. Ultrasonic diagnostic equipment has the following features that other modality tools do not provide: (1) real time analysis can be excellently made, (2) because of small size, use in operation rooms is rarely restricted and (3) not only used as a diagnosis tool but also used as a treatment tool. For the reason, the ultrasonic diagnostic equipment is further expected as a diagnosis-therapy integrated tool that can be used other than large hospitals.
To realize molecular imaging for “early diagnosis for a disease”, a site-specific oil-in-water emulsion using a liquid microbubble-precursor is conventionally known as an ultrasound contrast medium (for example, see Patent Literature 1).
The microbubble-precursor is a chemical substance that is easily gasified and can be stably present in a liquid state in the emulsion. A contrast medium, which produces micro air bubbles when ultrasound energy is applied thereto and has a size sufficient to travel from a blood vessel to a tissue, is proposed.
The microbubble precursor is administered into a living body in the form of nano-size liquid drops; however, liquid drops cause phase change by ultrasound irradiation to generate microbubbles. The microbubble precursor is, i.e., a phase-change ultrasound contrast medium. Owing to the ability of forming micro air bubbles by application of ultrasound energy, the contrast medium works in a site-specific manner. In addition, if a molecular probe such as antibody and ligand which selectively binds to a molecule constituting a living body is added, tissue selectivity can be provided.
Publication of Japanese Patent No. 3016592
Regarding the ultrasound contrast medium formed of a microbubble precursor disclosed in Patent Literature 1, the presence of the contrast medium in and out a cell cannot be determined; in particular, whether it is incorporated in a cell or not cannot be determined.
The invention provides a contrast medium whose uptake within a cell can be easily determined, a method and kit for preparing the contrast medium.
According to one aspect of the present invention, there is provided a contrast medium comprising at least one targeting molecular probe and at least one optical probe on a surface of a substrate having an ultrasound imaging function.
In the aspect, the contrast medium may have a first molecular probe for conjugation, a second molecular probe labeled with an optical probe and a third molecular probe for targeting capable of binding to a cell or a tissue in a disease-specific manner.
In the aspect, the third molecular probe may be labeled with the first molecular probe.
In the aspect, the second molecular probe and the third molecular probe may be contained in a molar ratio of 1:1.
In the aspect, the substrate may be a capsule-type contrast medium comprising a contrast substance having an imaging function included in an outer coat formed of lipid membrane.
In the aspect, the contrast substance may be a water insoluble substance vaporizable by ultrasound irradiation.
In the aspect, the contrast substance may be a gaseous material.
According to another aspect of the present invention, there is provided a method for preparing the contrast medium, comprising: a first step of producing a first solution containing the substrate of which the surface is labeled with the first molecular probe; a second step of producing a second solution containing the second molecular probe labeled with the optical probe; a third step of producing a third solution containing the third molecular probe labeled with the first molecular probe; a fourth step of producing a fourth solution by mixing the second solution produced in the second step and the third solution produced in the third step; a fifth step of mixing and reacting the first solution produced in the first step with the fourth solution produced in the fourth step with the first solution produced in the first step; and a sixth step of centrifuging the solution mixture produced in the fifth step.
In the aspect, the second molecular probe contained in the second solution and the third molecular probe contained in the third solution to be mixed in the fourth step may be present in a molar ratio of 1:1.
In the aspect, in the fifth step, a 10-fold or more dilution of the first solution produced in the first step may be mixed.
According to another aspect of the present invention, there is provided a kit for preparing the contrast medium as mentioned above, comprising: a plurality of storage containers separately storing a first solution containing the substrate of which a surface is labeled with the first molecular probe, a second solution containing the second molecular probe labeled with the optical probe, and a third solution containing the third molecular probe labeled with the first molecular probe; and openable and closable channels connected with the storage containers.
Contrast medium 1 according to a first embodiment of the present invention and a method for preparing the contrast medium will be described below with reference to the drawings.
The contrast medium 1 according to the embodiment has an ultrasound imageable substrate 2, at least one molecular probe (3 to 5) and at least one optical probe 6, which modify a surface of the substrate 2, as shown in
The substrate 2 is constituted of a liposome, which contains perfluoro hexane therein as an inclusive and has polyethylene glycol in the surface. Perfluoro hexane is a water insoluble substance, which is phase changeable into a gaseous material by irradiation with ultrasound at an intensity of a predetermined threshold or more. In other words, perfluoro hexane produces an ultrasound imaging function by changing a phase into gaseous material.
The molecular probes 3 to 5 include a first molecular probe 3 for conjugation to be attached to the surface of the substrate 2 as a label, a second molecular probe 4 to be attached to an optical probe 6 as a label and a third molecular probe 5 for targeting to be attached to the first molecular probe 3.
As the first molecular probe 3, for example, biotin is used.
As the second molecular probe 4, for example, streptavidin is used.
As the third molecular probe 5, for example, CCA1 antibody is used.
As the optical probe 6, for example, Alexa Fluor (AF) 647 is used.
A method for preparing the contrast medium 1 according to the embodiment having the constitution as mentioned above will be described below.
The method for preparing the contrast medium 1 of the embodiment, as shown in
To describe more specifically, in the first step S1, a first solution containing biotinylated nano liquid drops (biotin 3 is attached to the surface of the substrate 2 as a label) is produced. In the second step S2, a second solution containing streptavidin 4 labeled with AF647 6 is produced. In the third step S3, a third solution containing biotinylated CCA1 antibody prepared by labelling CCA1 antibody 5 with biotin 3 is produced.
In the fourth step S4, the fourth solution is produced by mixing the second solution (for example, 0.4 μM) and the third solution (for example, 0.4 μM) and allowing the resultant solution to stand still for 15 minutes for conjugation. In the fifth step S5, a dilution (for example, 20-fold) of the first solution (for example, 50 μL) is mixed with the fourth solution (for example, 50 μL) for a reaction, for example, at 4° C. for 30 minutes. In this manner, the surface of the substrate 2 of a contrast medium is simultaneously modified with CCA1 antibody and AF647 6.
In the sixth step S6, the solution mixture produced in the fifth step S5 is centrifuged for example, at 3000 G for 5 minutes. In this manner, the contrast medium 1 according to the embodiment is produced. In this step, a free composite not attached to the contrast medium 1 is separated and removed from the contrast medium.
Herein, it is important to carry out the third step S3 prior to the fourth step S4; mix the second solution and the third solution in a molar ratio of 1:1 in the fourth step S4; and mix a 10-fold or more dilution of the first solution with the fourth solution in the fifth step S5.
The concentration of the second solution can be roughly calculated, for example, by an absorption method. Furthermore, the concentration of the third solution can be accurately measured, for example, by use of an absorption method and Bradford method.
The action of the contrast medium 1 thus produced according to the embodiment will be described below.
The specific binding property of the contrast medium 1 according to the embodiment to large intestine cancer cells (DLD-1) cultured in a Petri dish was checked.
Herein, the supernatant centrifuged in the sixth step S6 was removed and the precipitate was resuspended in PBS (for example, 100 μL). The resultant solution was sprinkled on the cells and allowed to stand still for 30 minutes. The surface of the cells was washed with the culture solution.
The specific binding property was checked by a confocal fluorescence microscope and a flow-cytometer.
Confocal fluorescence microscope images are separately shown in
According to these figures, it is demonstrated that the contrast medium 1 according to the embodiment has a high accumulation level in the cell since it is labeled with CCA1 antibody serving as the third molecular probe 5. Since the contrast medium 1 according to the embodiment can be formed in the nano order, the contrast medium is bound to the cell surface if the culture time is short, whereas, the contrast medium is incorporated in the cell if the culture time is long. This phenomenon is called internalization. As a result, different functions can be shown as images with the passage of time from administration to a living body. Furthermore, the effect does not vary depending upon the component contained therein.
Furthermore, the contrast medium 1 according to the embodiment contains water insoluble perfluoro hexane therein as an inclusion. The perfluoro hexane is a substance, which causes phase change into a gaseous material by irradiation with ultrasound at an intensity of a predetermined threshold or more. The contrast medium 1 according to the embodiment labeled with an antibody and a pigment was subjected to functional evaluation as an ultrasound contrast medium, more specifically, evaluation of phase change from liquid to gas. One example of the results will be described with reference to
First, the water vessel 7 is filled with degassed water B of 37° C.
Then, a phantom (sample A) of a contrast medium according to the embodiment, which is contained in 8% acrylamide gel such that perfluoro pentane is contained in a concentration of 0.02 mg/mL, is placed in the water vessel 7. Pulse ultrasound of 3 MHz (duty ratio: 0.01, 1000 cycle) is applied to the phantom by use of the focused ultrasound transducer 8 for one second.
The ultrasound transducer 8 is driven by the wave generator 11 and the amplifier 10. During ultrasound irradiation by the ultrasound transducer 8, an ultrasound image of phantom A is obtained by an ultrasound probe 9 for diagnosis connected to the ultrasonic diagnostic equipment 12.
Note that even if the ultrasound irradiation condition from the focused ultrasound transducer 8 is changed to 9 MHz, the same results as in
Note that the same effect as in the embodiment was obtained in the case of a contrast medium using a low-boiling point water-insoluble substance in combination with a high-boiling point water-insoluble substance.
As described above, it was confirmed that even if an optical probe 6 and a molecular probe 5 such as CCA1 antibody are mounted on the surface of the substrate 2, vaporization can be made.
If a contract medium is prepared in accordance with the procedure as shown in
In the fifth step S5, if the first solution produced in the first step S1 is directly used without being diluted, a contrast medium is also agglomerated, as shown in
As described above, the contrast medium 1 and the preparation method for the contrast medium 1 according to the embodiment has an advantage in that the contrast medium is allowed to be specifically bound only to a target molecule of a living body (cell) by means of the third molecular probe 5 without agglomerating particles of the contrast medium 1; and also has another advantage in that imaging of the state (function) of a living body, which varies with time after administration of the contrast medium, can be made by light and ultrasound.
As a result, using the fluorescence imaging by the contrast medium 1 according to the embodiment, it is possible to previously understand behavior of the contrast medium 1 according to the embodiment in the cell such as timing of internalization. It is also possible to perform ultrasound irradiation at an optimal time while observing fluorescence in real time.
To be more specific, it is possible to provide a contrast medium having sensitivity to not only ultrasound but also light, which is excellent in real-time imaging and resolution, and having a size sufficient to travel into tissues through blood vessel by labeling the ultrasound imageable substrate 2 with the molecular probe 3 and the optical probe 6. Owing to this, the contrast medium has an advantage of early diagnosing a disease at a cellular level.
It is also possible to appropriately set the timing for vaporizing the contrast medium by ultrasound irradiation. More specifically, imaging of various functions can be made also by ultrasound irradiation if the contrast medium 1 according to the embodiment is used.
In the embodiment, perfluoro hexane, which is a water insoluble substance vaporizable by ultrasound irradiation, is used as an inclusion in the contrast medium 1; however, other water insoluble substances may be used, or alternatively, water insoluble substances different in boiling point may be used in combination. Besides this, contrast substances sensitive to other modality means may be contained in the contrast medium in order to use other modality means except ultrasound and light, in combination.
The molecular probes 3 to 5 and the optical probe 6 are not limited to those used in the embodiment.
In the embodiment, a nano-size bubble liposome, which is prepared by using a solution containing perfluoro butane as an inclusion in place of perfluoro hexane and lipid membrane formed of polyethylene glycol as an outer coat, may be employed.
When the contrast medium 1 thus prepared is injected to a mouse tail, the contrast medium can be selectively accumulated only to a tumor tissue, as shown in
Examples of the inclusion of the contrast medium 1 may include air and gaseous materials such as argon, perfluoro butane and perfluoro propane. The material for the outer coat is not limited to the lipid membrane formed of polyethylene glycol and lipid materials having other hydrophilic groups may be used. Furthermore, if information on internalization is not required, a step of labeling the contrast medium with an optical dye may be skipped.
Furthermore, since the contrast medium according to the embodiment has tissue selectivity to a cancer cell and an internalization ability to enter into the cell, the contrast medium can serve as a delivery means for any cancer cell. The structure of the delivery means may be the same as that of the contrast medium according to the embodiment. The delivery means may be, for example, a substrate binding to a targeting molecular probe via a conjugate molecular probe having optical activity.
The substrate may contain a desired substance having an anti-cancer function. Alternatively, a substance, which is activated only upon application of therapeutic-purpose ultrasound, may be contained. The delivery means thus constituted may be used, for example, as diagnostic/therapeutic agent.
Now, the method for preparing a contrast medium according to a second embodiment of the present invention will be described below with reference to the drawings.
The contrast medium according to the embodiment employs a mixture of two types of gaseous materials as an inclusion, more specifically, a gas mixture of perfluoro hexane and perfluoro pentane contained in a ratio of 1:1.
The size of the contrast medium according to the embodiment thus constituted is compared to those of the contrast mediums obtained by other preparation methods as shown in Table 1. Table 1 shows the results of two experimental results.
If streptavidin (SA) is singly added to the first solution produced in the first step, the size of the contrast medium considerably increases and agglomerated; however, if the contrast medium is prepared by the preparation method according to the embodiment, the medium can be labeled with an antibody while maintaining a nano-size. The mixing ratio of the water insoluble substances is not limited to those of the embodiment. The type of inclusions and combination and mixing ratio thereof, can be appropriately changed depending upon the vaporization conditions (size of air bubbles, retention time of vapor phase, frequency responsiveness and ultrasound intensity threshold).
As described above, the contrast medium produced by the preparation method according to the embodiment, even if a gaseous material serving as an inclusion is changed, causes no agglomeration of particles of the contrast medium and specifically bind only to a target molecule of a living body (cell) by means of the third molecular probe. Further, imaging of the state (function) of a living body, which varies with time after administration of the contrast medium, can be made.
Herein, specific binding property to HCT116 cell and AGS cell in place of DLD1 cell was checked, without changing CCA1 antibody serving as the third molecular probe 5.
To describe it more specifically, the contrast medium was added to culture dishes of HCT116 cell and AGS cell and allowed to stand still, for example, one hour, and the surface are washed with the culture medium. For example, three hours later, cell-specific uptake was observed by a confocal laser microscope. The cells were cultured for 12 hours, washed with PBS, removed from the culture dishes with trypsin/EDTA and subjected to flow-cytometric analysis.
Accumulation results are shown in
The contrast medium according to the embodiment was also accumulated in HCT116 cell and travels into the cell; however, the contrast medium was not accumulated in AGS cell, whose expression level of CCA1 is reported to be low.
As described above, the contrast medium according to the embodiment can specifically bind to the antigen corresponding to the antibody employed as the label and advantageously distinguish the cell from other cells.
In the present invention, the contrast medium 1 can be produced by a preparation kit 21 shown in
The preparation kit 21 has storage containers 22, 23 and 24 for aseptically storing first to third solutions, respectively, and channels 25 and 26 connecting between these storage containers 22 to 24. The channels 25, 26 are equipped respectively with valves 27 and 28, which are operated to open and close the channels 25 and 26.
The second solution and the third solution are mixed by opening the valve 28 of the second channel 26 to produce the fourth solution, and then the first solution and the fourth solution are mixed by opening the valve 27 of the first channel 25. The contrast medium 1 according to the embodiment can be aseptically produced by setting the whole preparation kit in a centrifuge. Note that a safety apparatus may be provided such that the valve 28 and valve 27 are opened only in this order.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2013016610 | Jan 2013 | JP | national |
This is a continuation of International Application PCT/JP2014/051311, with an international filing date of Jan. 22, 2014, which is hereby incorporated by reference herein in its entirety. This application claims the benefit of Japanese Patent Application No. 2013-016610, the content of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2014/051311 | Jan 2014 | US |
| Child | 14812456 | US |
