Method for producing a platinum nanoparticle-containing composition

Abstract

To provide a method for producing a platinum nanoparticle-containing composition that is composed of a single material and has both of fluorescence properties and magnetic properties in the near-infrared region.SOLUTION: This method includes: a bonding step 100 for reacting a platinum ion 20 with the 5th to 7th generation polyamide amine dendrimer 10 under ice-cold conditions to form a chemical bond between the platinum ion 20 and the polyamide amine dendrimer 10, incorporating the platinum ion 20 into the polyamide amine dendrimer 10; a reduction step 200 for reducing the polyamide amine dendrimer 10 having incorporated the platinum ion 20 with fructose, to synthesize a platinum nanoparticle 30 in the polyamide amine dendrimer 10; and a modification step 300 for reacting the platinum nanoparticle 30 with GAVA 50, bonding the platinum nanoparticle 30 to an amino group 40.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for making platinum nanoparticle-containing compositions that can be used as fluorescent and magnetic probes. [Background technology]

[0002] Fluorescent probes using nanoparticles (metal nanoparticles and the like) are being developed in various fields such as nanomaterials, optoelectronics, and medicine (see, for example, Patent Documents 1 and 2). In particular, in the medical field, fluorescent probes are indispensable for research and treatment because they are capable of detecting cancer cells with high sensitivity by reacting with or binding to specific target molecules, such as cancer cells.

[0003] The invention described in Patent Document 1 involves producing silicon by heating and refluxing silicon chloride or the like in a solvent containing dimethylformamide. This manufacturing method does not require the use of highly hazardous reagents or post-treatment, and furthermore allows for the mass production of nanoparticle fluorescent materials with uniform particle sizes.

[0004] Moreover, the invention described in Patent Document 2 involves reducing a metal compound in an aqueous solution that contains a protein and has an adjusted pH, to produce a phosphor composition. According to this manufacturing method, it is possible to mass-produce phosphor compositions containing metal nanoparticles without using highly hazardous reagents in the manufacturing process. It is also possible to produce a wide range of phosphor compositions containing various metal nanoparticles with different controlled particle sizes.

[0005] Here, for fluorescent probes to be used in the medical field, it is of course essential that they are not toxic to living organisms. However, the invention described in Patent Document 1 is a synthesis method that is carried out in an organic solvent in which living organisms cannot survive, and is therefore not suitable as a fluorescent probe for the medical field. Furthermore, since there are various molecules in the living body that absorb visible light, light with a wavelength of approximately 400 to 570 nm is absorbed by molecules on the skin surface and within the living body, making it impossible to observe fluorescent signals of this wavelength from deep within the living body. Therefore, in order to establish high-definition medical diagnostic techniques for cancer diagnosis, stem cell therapy, and the like, a fluorescent probe must have an emission wavelength in the near-infrared region (600 to 850 nm) that is not absorbed or scattered by biological tissue and can be observed even from deep within the living body. However, the invention described in Patent Document 1 is not a phosphor that has fluorescent properties in the near-infrared region.

[0006] Furthermore, fluorescent proteins such as those described in Patent Document 2 fade, that is, lose their properties as fluorescent substances, when used for observation for several minutes to an hour. Even if these fluorescent substances are stored under conditions that minimize fading (in a dark place at 4°C), their optical properties (brightness (luminance)) will drop to less than half in about one or two months. In other words, it is difficult to use this fluorescent probe for the examination and diagnosis of cancer metastasis, which requires long-term follow-up.

[0007] Therefore, the present inventors have provided a metal nanoparticle-containing composition that has fluorescent properties in the near-infrared region, has low toxicity to living organisms, and does not fade for a long period of time, by using a production method that includes a bonding step in which platinum ions are reacted with a fifth- to seventh-generation polyamidoamine dendrimer at ice-cooling temperature to form a chemical bond between the platinum ions and the polyamidoamine dendrimer, thereby incorporating platinum ions into the polyamidoamine dendrimer, and a reduction step in which platinum nanoparticles are synthesized within the polyamidoamine dendrimer 10 by reducing the polyamidoamine dendrimer that has incorporated platinum ions with fructose (Patent Document 3).

[0008] In this way, bioimaging methods that use light can capture not only the temporal and quantitative dynamics and localization of target molecules, but also non-invasively and continuously visualize not only molecular expression and function, but also the intracellular environment and stress. Therefore, molecular imaging using light can be used to make detailed assessments of not only cancer onset and metastasis, but also cell function information and accumulation in tissues during stem cell differentiation.

[0009] However, optical measurement technology has difficulty obtaining whole-body images, making it difficult to simultaneously diagnose the physiological information and location within the body of cancer cells and stem cells. Therefore, it is difficult to diagnose the presence or absence of cancer, as well as its exact shape and location within the body, without taking into account anatomical information from whole-body images obtained by magnetic resonance imaging (MRI).

[0010] Therefore, the present inventors focused on establishing a multimodal imaging measurement method that combines fluorescence imaging and MRI using a single molecular probe. It is known that fluorescent silica-based nanoparticles have been proposed that enable accurate detection, characterization, monitoring and treatment of diseases such as cancer (Patent Document 4). Patent Document 4 describes that ions having magnetic properties can be conjugated to fluorescent silica-based nanoparticles, but the details are not clear, and at least it is not a single material. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent No. 5452098 [Patent Document 2] JP 2012-246449 A [Patent Document 3] Patent No. 6573370 [Patent Document 4] Special Publication No. 2016-516729 Summary of the Invention [Problem to be solved by the invention]

[0012] An object of the present invention is to provide a method for producing a platinum nanoparticle-containing composition that simultaneously has fluorescent properties in the near infrared region and magnetic properties from a single material. [Means for solving the problem]

[0013] In order to achieve the above object, the method for producing a platinum nanoparticle-containing composition (1) of the present invention includes a bonding step (100) of reacting platinum ions (20) with a fifth to seventh generation polyamidoamine dendrimer (10) under ice cooling to form a chemical bond between the platinum ions (20) and the polyamidoamine dendrimer (10), thereby incorporating the platinum ions (20) into the polyamidoamine dendrimer (10); a reduction step (200) of synthesizing platinum nanoparticles (30) within the polyamidoamine dendrimer (10) by reducing the polyamidoamine dendrimer (10) having the platinum ions (20) incorporated therein with fructose; GABA The method is characterized by comprising a modification step (300) in which an amino group (40) is bonded to the platinum nanoparticles (30) by reacting with the above.

[0014] The present invention also relates to the GABA is characterized as being 4-aminobutyric acid.

[0015] The present invention is also characterized by comprising a binding step (100) of incorporating platinum ions (20) into the polyamidoamine dendrimer (10) by reacting platinum ions (20) with a fifth to seventh generation polyamidoamine dendrimer (10) under ice cooling to form a chemical bond between the platinum ions (20) and the polyamidoamine dendrimer (10); a reduction step (200) of synthesizing platinum nanoparticles (30) within the polyamidoamine dendrimer (10) by reducing the polyamidoamine dendrimer (10) having the platinum ions (20) incorporated therein with fructose; and a modification step (300) of reacting the platinum nanoparticles (30) with β-alanine to bind amino groups (40) to the platinum nanoparticles (30).

[0016] The present invention is also characterized in that the obtained platinum nanoparticle-containing composition (1) is isolated and purified.

[0017] Here, the symbols in parentheses indicate corresponding elements or items shown in the drawings and in the detailed description below. Effect of the Invention

[0018] According to the present invention, the produced platinum nanoparticle-containing composition has fluorescent properties in the near-infrared region, so the emitted light is unlikely to be absorbed or scattered by biological tissues. Therefore, the fluorescence of the platinum nanoparticle-containing composition can be observed with a resolution of 1 μm or less and high sensitivity even from deep inside the body (several mm to several tens of cm). In addition, since the platinum nanoparticle-containing composition has magnetic properties, by combining it with MRI, which can measure the whole body, it is possible to obtain whole-body images of a living body, establishing a multimodal image measurement method.

[0019] This allows us to pinpoint the exact location of cancer at the single-cell level, making it possible to diagnose early cancer and cancer metastasis.In addition, it will be possible to extract highly accurate bioimage information containing molecular information at various scales, from individuals to cells, and will dramatically advance research on cancer treatment and regenerative medicine, such as observing the molecular mechanisms of cancer expression and cancer metastasis in vivo, elucidating the differentiation mechanisms of cancer cells, and tracking them in vivo.

[0020] In addition, since reduction is performed using fructose, a weak reducing agent, at a mild temperature of 80 to 90° C., the formed platinum nanoparticles 30 have low cytotoxicity. In particular, since the platinum nanoparticle-containing composition has a small size of 1.1 nm to 1.5 nm, it is difficult for the composition to accumulate in cells even if it is left in the body for a long period of time, and cytotoxicity due to metal accumulation is extremely low. In addition, since the platinum used in the synthesis is stable and difficult to oxidize, the risk of ionization due to oxidation reactions in the body and of biotoxicity due to the generated metal ions is lower than that of metal nanoparticles made from other metal materials (iron, cobalt, palladium, nickel, etc.).

[0021] Such low toxicity means that it has little impact on not only living organisms but also the environment. The platinum nanoparticle-containing composition according to the present invention can also be applied to solar cells (solar panels). Sunlight contains about 50% visible light and about 30% near-infrared light. When the platinum nanoparticle-containing composition according to the present invention is used in solar cells, near-infrared light, which is hardly used by existing solar cells, can also be used for power generation, so that the power generation efficiency can be dramatically improved. Furthermore, when the platinum nanoparticle-containing composition according to the present invention is used in solar cells, etc., the impact on the environment and human body is small even if optical materials leak due to failure or damage. Furthermore, because the platinum nanoparticle-containing composition is chemically stable, it can be used semi-permanently as an optical material.

[0022] Even when used for long-term observation, the luminance (brightness) is maintained without fading, and the optical properties are maintained even when the platinum nanoparticle-containing composition is stored at room temperature for more than six months, so it can be used for examinations and diagnoses that require follow-up observation. For example, it is useful for examinations of cancer metastasis. In addition, by labeling stem cells and observing them for a long period of time after transplantation into living tissue, it is possible to evaluate the behavior (migration) of stem cells in the body and the processes of differentiation, proliferation, regeneration, etc., and therefore it is possible to visualize and diagnose the regeneration and treatment processes of living tissues by stem cells.

[0023] Incidentally, the above-mentioned Patent Documents 1 to 4 do not disclose at all a method for producing a platinum nanoparticle-containing composition that simultaneously has fluorescent properties and magnetic properties in the near-infrared region from a single material, as in the present invention. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic diagram showing a bonding step 100 of a method for producing a platinum nanoparticle-containing composition according to an embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic diagram showing a reduction step 200 of a method for producing a platinum nanoparticle-containing composition according to an embodiment of the present invention. [Diagram 3]FIG. 3 is a schematic diagram showing a modification step 300 of a method for producing a platinum nanoparticle-containing composition according to an embodiment of the present invention. [Figure 4] 1 is a graph showing the fluorescence properties of a platinum nanoparticle-containing composition according to an embodiment of the present invention. [Diagram 5] 1 is a graph showing magnetic properties of a platinum nanoparticle-containing composition according to an embodiment of the present invention, in which 4-aminobutyric acid, a type of GABA, was added at a molar ratio of 100 times that of platinum nanoparticles 30. [Figure 6] 1 is a graph showing magnetic properties of a platinum nanoparticle-containing composition according to an embodiment of the present invention, in which 4-aminobutyric acid, a type of GABA, was added at a molar ratio of 50 times that of platinum nanoparticles at 30. [Figure 7] 1 is a graph showing magnetic properties of a platinum nanoparticle-containing composition according to an embodiment of the present invention, in which β-alanine was added in an amount 100 times the amount of platinum nanoparticles (30) based on a molar ratio. [Figure 8] 1 is a graph showing magnetic properties of a platinum nanoparticle-containing composition according to an embodiment of the present invention, in which β-alanine was added in an amount of 50 times the molar ratio of platinum nanoparticles (30). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] A method for producing a platinum nanoparticle-containing composition 1 according to an embodiment of the present invention will be described with reference to FIGS. The method for producing the platinum nanoparticle-containing composition 1 includes a binding step 100, a reduction step 200, a modification step 300, and an isolation step.

[0026] In the bonding step 100, 5 mL of ultrapure water was added to a glass screw tube (10 mL), and then 90 μL of hexachloroplatinic acid (IV) hexahydrate (H2PtCl6·6H2O) (0.5 M) was added to 0.25 μmol of the template molecule, fifth-generation polyamidoamine dendrimer 10 (PAMAM G5-OH), under ice cooling (4°C), and the mixture was allowed to react for 5 days. By carrying out the reaction at such a low temperature, as shown in FIG. 1, a larger amount of platinum ions 20 are incorporated into the polyamidoamine dendrimer 10, thereby increasing the efficiency of synthesis in the next reduction step 200.

[0027] Next, in the reduction step 200, fructose (D-fructose) adjusted to a concentration of 1M was added to platinum ions 20 at a molar ratio of 50 times (50:1), and the reduction reaction was carried out at a temperature of 80 to 90°C for two weeks. As a result, platinum nanoparticles 30 are synthesized within the polyamidoamine dendrimer 10 as shown in FIG.

[0028] Next, in the modification step 300, at room temperature (20 to 25° C.), GABA Among 50 4-Aminobutyric Acid was added to platinum nanoparticles 30 at a molar ratio of 100:1, and the reaction was allowed to proceed in a cool, dark place for one week. As a result, a platinum nanoparticle-containing composition 1 is obtained in which an amino group (NH2) is bound around the platinum nanoparticle 30, as shown in Fig. 3. The molar concentration of the platinum nanoparticle-containing composition 1 was 324.9 pmol (picomole).

[0029] Finally, in the isolation step, impurities are removed using an ultracentrifuge and a high performance liquid chromatograph (HPLC) equipped with a near-infrared fluorescence detector, and the platinum nanoparticle-containing composition 1 is isolated and purified.

[0030] When the platinum nanoparticle-containing composition 1 thus produced was irradiated with light having a wavelength of 535 nm as excitation light, red fluorescence (near-infrared light) having an emission wavelength of 630 nm was observed, as shown in FIG. It was also found that the platinum nanoparticles 30 according to this embodiment have a particle size of 1.1 to 1.5 nm and a quantum yield of 0.5%.

[0031] When the platinum nanoparticle-containing composition 1 was produced using the first to third generation polyamidoamine dendrimers 10, the platinum nanoparticle-containing composition 1 did not exhibit fluorescence. On the other hand, when the platinum nanoparticle-containing composition 1 was produced using the fourth-generation polyamidoamine dendrimer 10, it had green fluorescence (emission wavelength: 520 nm).

[0032] Generally, the smaller the particle radius of metal nanoparticles, the more the emission wavelength shifts to the blue (shorter wavelength) side. In addition, the particle size of metal nanoparticles can be increased by using a dendrimer of a larger generation. From the above results, it can be seen that platinum nanoparticle-containing composition 1 produced using polyamidoamine dendrimer 10 of fifth generation or higher has red fluorescence. Here, it is known that the particle diameter of conventional red-glowing metal nanoparticles is about 2.3 nm, but it was also found that the platinum nanoparticles 30 according to this embodiment are about half this size.

[0033] Furthermore, when a magnetic field was applied to the produced platinum nanoparticle-containing composition 1, it was observed that platinum nanoparticle-containing composition 1 was magnetically polarized and magnetized into positive and negative poles, as shown in Figure 5. The horizontal axis indicates the magnetic field, while the vertical axis indicates the magnetization, which is normalized to be approximately 1 when saturated. Also, what is shown in FIG. 6 is a modification process 300, GABA In 4-Aminobutyric Acid was added to platinum nanoparticles 30 in a molar ratio of 50 times (50:1), and it can be seen that platinum nanoparticle-containing composition 1 is clearly magnetized. The molar concentration of platinum nanoparticle-containing composition 1 was 195.8 pmol (picomole).

[0034] This confirmed that the produced platinum nanoparticle-containing composition 1 was a single material that simultaneously had fluorescent properties in the near infrared region and magnetic properties.

[0035] Furthermore, it was confirmed that the platinum nanoparticle-containing composition 1 is useful as a fluorescent probe and a magnetic probe for cancer cells, and it was also confirmed that at the concentration of platinum nanoparticles 30 required to function as this fluorescent probe and magnetic probe, the toxicity of the platinum nanoparticles 30 is sufficiently low that they can be used in living organisms.

[0036] The platinum nanoparticle-containing composition 1 produced as described above has fluorescent properties in the near infrared region, so the emitted light is unlikely to be absorbed or scattered by biological tissues. Therefore, the fluorescence of the platinum nanoparticle-containing composition 1 can be observed with a resolution of 1 μm or less and high sensitivity even from deep inside the body (several mm to several tens of cm). In addition, since the platinum nanoparticle-containing composition 1 has magnetic properties, by combining it with MRI, which can measure the entire body, it is possible to obtain whole-body images of a living body, establishing a multimodal image measurement method.

[0037] This allows us to pinpoint the exact location of cancer at the single-cell level, making it possible to diagnose early cancer and cancer metastasis.In addition, it will be possible to extract highly accurate bioimage information containing molecular information at various scales, from individuals to cells, and will dramatically advance research on cancer treatment and regenerative medicine, such as observing the molecular mechanisms of cancer expression and cancer metastasis in vivo, elucidating the differentiation mechanisms of cancer cells, and tracking them in vivo.

[0038] In addition, since reduction is performed using fructose, a weak reducing agent, at a mild temperature of 80 to 90° C., the formed platinum nanoparticles 30 have low cytotoxicity. In particular, since the platinum nanoparticle-containing composition 1 according to this embodiment has a small size of 1.1 nm to 1.5 nm, it is difficult to accumulate in cells even if it is present in a living body for a long period of time, and cytotoxicity due to metal accumulation is extremely low. In addition, since the platinum used in the synthesis is stable and difficult to oxidize, the risk of ionization due to oxidation reactions in the body and of biotoxicity due to the generated metal ions is lower than that of metal nanoparticles made from other metal materials (iron, cobalt, palladium, nickel, etc.).

[0039] Furthermore, even when used for long-term observation, the luminance (brightness) is maintained without fading, and the platinum nanoparticle-containing composition 1 maintains its optical properties even when stored at room temperature for more than six months, so it can be used for examinations and diagnoses that require follow-up observation. For example, it is useful for examinations of cancer metastasis. In addition, by labeling stem cells and observing them for a long period of time after transplantation into living tissue, it is possible to evaluate the behavior (migration) of stem cells in the body and the processes of differentiation, proliferation, regeneration, etc., and therefore it is possible to visualize and diagnose the regeneration and treatment processes of living tissues by stem cells.

[0040] In this embodiment, in the modification step 300, at room temperature (20 to 25° C.), GABA In 4-Aminobutyric Acid was added to platinum nanoparticles 30 in a molar ratio of 100:1, and then reacted in a cool, dark place for one week. 4-Aminobutyric Acid Instead of β-alanine, β-alanine can be used. That is, in the modification step 300, β-alanine can be added at room temperature (20 to 25°C) so that the molar ratio is 100 times (100:1) relative to the platinum nanoparticles 30, and then the mixture can be reacted in a cool, dark place for one week. This also makes it possible to obtain a platinum nanoparticle-containing composition 1 in which an amino group (NH2) is bound around the platinum nanoparticles 30. The molar concentration of the platinum nanoparticle-containing composition 1 was 94.3 pmol (picomole).

[0041] In this case, when a magnetic field was applied to platinum nanoparticle-containing composition 1 produced using β-alanine, it was observed that platinum nanoparticle-containing composition 1 was magnetized, polarized to the positive and negative poles, although the amount was very small, as shown in Figure 7. The horizontal axis shows the magnetic field, while the vertical axis shows magnetization in the form of raw data. 8 shows that β-alanine was added in the modification step 300 at a molar ratio of 50 times (50:1) the platinum nanoparticles 30, and it can be seen that the platinum nanoparticle-containing composition 1 is magnetized, although the amount is very small. The molar concentration of the platinum nanoparticle-containing composition 1 was 36.4 pmol (picomolar).

[0042] As a result, the platinum nanoparticle-containing composition 1 produced using β-alanine was GABA In 4-Aminobutyric Acid It was confirmed that a single material has both fluorescent properties in the near infrared region and magnetic properties, similar to the platinum nanoparticle-containing composition 1 produced using the method described above. GABA In 4-Aminobutyric Acid The platinum nanoparticle-containing composition had the same effect as that of the platinum nanoparticle-containing composition 1 produced using the method described above.

[0043] In the present embodiment, the reduction step 200 is carried out at a temperature of 80 to 90°C, but the temperature is not limited to this and may be any temperature within the range of 70 to 90°C. [Explanation of symbols]

[0044] 1. Platinum nanoparticle-containing composition 10 Polyamidoamine Dendrimer 20 Platinum ion 30 Platinum Nanoparticles 40 Amino group 100 Bonding process 200 Reduction process 300 Modification process

Claims

1. a bonding step of reacting platinum ions with a fifth to seventh generation polyamidoamine dendrimer under ice cooling to form a chemical bond between the platinum ions and the polyamidoamine dendrimer, thereby incorporating the platinum ions into the polyamidoamine dendrimer; a reduction step of synthesizing platinum nanoparticles within the polyamidoamine dendrimer by reducing the polyamidoamine dendrimer having the platinum ions incorporated therein with fructose; A method for producing a platinum nanoparticle-containing composition, comprising a modification step of reacting the platinum nanoparticles with GABA to bond amino groups to the platinum nanoparticles.

2. The method for producing a platinum nanoparticle-containing composition according to claim 1, wherein the GABA is 4-aminobutyric acid.

3. a bonding step of reacting platinum ions with a fifth to seventh generation polyamidoamine dendrimer under ice cooling to form a chemical bond between the platinum ions and the polyamidoamine dendrimer, thereby incorporating the platinum ions into the polyamidoamine dendrimer; a reduction step of synthesizing platinum nanoparticles within the polyamidoamine dendrimer by reducing the polyamidoamine dendrimer having the platinum ions incorporated therein with fructose; A method for producing a platinum nanoparticle-containing composition, comprising a modification step of reacting the platinum nanoparticles with β-alanine to bind amino groups to the platinum nanoparticles.

4. A method for producing a platinum nanoparticle-containing composition, comprising isolating and purifying the platinum nanoparticle-containing composition obtained according to any one of claims 1 to 3.

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