NEAR INFRARED ABSORBING DYE-BASED COMPOSITE PARTICLES EXHIBITING PHOTOTHERMAL EFFECT, METHOD FOR MANUFACTURING THE SAME, AND USE THEREOF

Abstract

The present invention relates to near-infrared-absorbing dye-based composite particles which exhibit a photothermal effect and/or photoacoustic signal upon photoirradiation, a preparation method thereof, and a use thereof. The near-infrared-absorbing composite particles comprise: a water-insoluble salt of a near-infrared-absorbing dye, which comprises anions of the near-infrared-absorbing dye and metal cations capable of forming a precipitation product with the anions of the near-infrared-absorbing dye; and particles of a polymeric surfactant, in which a water-insoluble salt of the near-infrared-absorbing dye is supported in the hydrophobic part of the polymeric surfactant.

Claims

1. A method for preparing a near-infrared-absorbing dye-based composite particle, comprising: a first step, in which an ionizable near-infrared-absorbing dye, that is in a state dissolved in an aqueous medium, undergoes a precipitation reaction with an ionic compound capable of providing a metal cation that is able to form a precipitation product with an anion of the near-infrared-absorbing dye, and forms a water-insoluble salt of the near-infrared-absorbing dye; and a second step, in which a dispersion in which the water-insoluble salt of the near-infrared-absorbing dye is dispersed is mixed with an aqueous solution comprising a polymeric surfactant and then supports the water-insoluble salt of the near-infrared-absorbing dye in a particle of the polymeric surfactant.

2. The method of claim 1, wherein the second step not only supports the water-insoluble salt of the near-infrared-absorbing dye in the particle of the polymeric surfactant, but also further supports, in the particle of the polymeric surfactant, an additive selected from the group consisting of a therapeutic agent, a diagnostic agent, and a contrast agent.

3. The method of claim 1, wherein, in the first step, the metal cation that is able to form a precipitation product with an anion of a near-infrared-absorbing dye is an iron ion.

4. The method of claim 1, wherein, in the first step, the ionizable near-infrared-absorbing dye is at least one selected from the group consisting of a hydrophilic cyanine dye comprising a sulfonate group, indocyanine green (ICG), cyanine 5.5 (Cy5.5), and cyanine 7 (Cy7).

5. The method of claim 1, wherein the near-infrared-absorbing dye-based composite particle is a near-infrared-absorbing composite particle that exhibits a photothermal effect or photoacoustic signal upon photoirradiation.

6. The method of claim 1, wherein the near-infrared-absorbing dye-based composite particle being prepared exhibits inhibition of non-specific aggregation, inhibition of absorption into serum proteins, inhibition of photodegradation, or a combination thereof, compared to the ionizable near-infrared-absorbing dye of the first step.

7. A near-infrared-absorbing composite particle comprising: a water-insoluble salt of a near-infrared-absorbing dye, which comprises: an anion of the near-infrared-absorbing dye; and a metal cation capable of forming a precipitation product with the anion of the near-infrared-absorbing dye; and a particle of a polymeric surfactant, in which a water-insoluble salt of the near-infrared-absorbing dye is supported in the hydrophobic part of the polymeric surfactant, wherein the near-infrared-absorbing composite particle exhibits a photothermal effect photoacoustic signal or both upon photoirradiation.

8. The near-infrared-absorbing composite particle of claim 7, wherein the near-infrared-absorbing dye is indocyanine green (ICG) and the metal cation is an iron ion.

9. The near-infrared-absorbing composite particle of claim 7, wherein the composite particle is a nanoparticle that is able to be dispersed as a colloidal particle in a bodily fluid.

10. The near-infrared-absorbing composite particle of claim 7, wherein the polymeric surfactant has a molecular weight of 7,500 to 15,000.

11. The near-infrared-absorbing composite particle of claim 7, wherein the particle of the polymeric surfactant further supports an additive selected from the group consisting of a therapeutic agent, a diagnostic agent, and a contrast agent is further supported in.

12. The near-infrared-absorbing composite particle of claim 7, wherein the anion of the near-infrared-absorbing dye is contained in an amount of 0.4 to 40 parts by weight, the metal cation in an amount of 0.1 to 10 parts by weight, and the polymeric surfactant in an amount of 50 to 99.5 parts by weight, relative to 100 parts by weight of the near-infrared-absorbing composite particle.

13. The near-infrared-absorbing composite particle of claim 7, wherein the near-infrared-absorbing composite particle is a heat source that is able to be heated to a temperature of 45 C. or higher through the photothermal effect.

14. The near-infrared-absorbing composite particle of claim 7, wherein the near-infrared-absorbing composite particle is prepared by the method of claim 1.

15. A method for using the near-infrared-absorbing composite particle of claim 7 as a photothermal therapeutic agent, a diagnostic agent, an imaging or mapping probe, a drug carrier, or a combination thereof that absorbs light in the near-infrared region and generates heat.

16. A method for photothermal treatment, comprising: (a) administering the near-infrared-absorbing composite particle of claim 7 to a patient; and (b) applying photoirradiation to allow the near-infrared-absorbing composite particle to absorb light in the near-infrared region and generate heat.

17. The method of claim 16, wherein step (a) is a step to administer the near-infrared-absorbing composite particle to the blood vessel of a patient to be accumulated in a cancer cell, and step (b) is a step to perform a photothermal treatment of the cancer cell via photoirradiation.

18. A method for realizing photothermal or photoacoustic imaging or mapping, comprising: (a) administering the near-infrared-absorbing composite particle of claim 7 to a subject; and (b) applying photoirradiation to allow the near-infrared-absorbing composite particle to absorb light in the near-infrared region and generate heat and thereby provide a photothermal or photoacoustic signal or image.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0069] FIG. 1 shows a chemical structure of a near-infrared fluorescent dye, indocyanine green (ICG), according to an embodiment of the present invention; and a schematic diagram illustrating the design of finally prepared nanoparticles in which ICG is encapsulated.

[0070] FIG. 2 shows (a) an image illustrating the formation of a hydrophobic ICG-Fe composite according to an embodiment of the present invention; and (b) the results of the formation confirmed by the absorption/fluorescence spectrum.

[0071] FIG. 3 shows (a) a transmission electron microscope (TEM) image and (b) the results of the absorption/fluorescence spectrum of ICG-Fe-encapsulated nanoparticles (ICG-Fe NPs) including a hydrophobicized ICG-Fe composite according to an embodiment of the present invention.

[0072] FIG. 4 shows (a) a fluorescence image illustrating the ICG-Fe NPs according to an embodiment of the present invention with regard to their colloidal stability in a biomimetic environment via a fluorescence imaging device (IVIS); and (b) a graph illustrating the intensity of the fluorescence signals detected therefrom.

[0073] FIG. 5 shows the evaluation results of the ICG-Fe NPs according to an embodiment of the present invention with regard to their photostability in the presence of light, in which (a) shows an image illustrating changes in color; and (b) shows graphs illustrating the degree of changes in relative absorption/fluorescence signals, after photoirradiation, respectively.

[0074] FIG. 6 shows a graph illustrating the photothermal properties by comparing the degree of a temperature increase of the ICG-Fe NPs according to an embodiment of the present invention.

[0075] FIG. 7 shows an image illustrating the photoacoustic image signals of the ICG-Fe NPs according to an embodiment of the present invention.

[0076] FIG. 8 shows the results of confirming the presence/absence of in vivo toxicity of the ICG-Fe NPs according to an embodiment of the present invention by (a) blood liver levels and (b) a liver/kidney tissue staining method.

[0077] FIG. 9 shows images illustrating the presence/absence of a characteristic of accumulation in cancer cells via fluorescence imaging in a cancer model mouse administered with the ICG-Fe NPs according to an embodiment of the present invention. The red arrows indicate the location of the cancer tissues.

[0078] FIG. 10 shows images illustrating the effect of photothermal therapy in a cancer model mouse administered with the ICG-Fe NPs according to an embodiment of the present invention. The red arrows indicate the location of the cancer tissues.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Preparation of ICG-Encapsulated Nanoparticles

[0079] The ICG-encapsulated nanoparticles according to the present invention were prepared, for example, such that the ICG, which shows absorption/fluorescence in the near-infrared region and simultaneously has a photothermal effect, can be present inside of the polymer nanoparticles in a stable state and have improved optical characteristics. The compositions of these particles and the specific preparation methods are as follows.

Example 1-1. Preparation of Hydrophobic ICG-Fe Composite which can be Loaded into Hydrophobic Inside of Polymer Nanoparticles

[0080] 20 mg of indocyanine green (ICG, MW: 774.96, Tokyo Chemical Industry, Japan) and 30 mg of iron(III) chloride (FeCl.sub.3, MW: 162.2, Sigma-Aldrich, USA) were each dissolved in 20 mL of water, and then the two solutions were mixed together. The mixture was reacted at room temperature for 1 hour, and the supernatant was removed when the precipitation product had sedimented. The precipitated ICG-Fe composite was washed with an excess amount of water. The purified ICG-Fe composite was dried at room temperature under vacuum. The thus-prepared ICG-Fe composite was used to prepare nanoparticles in Example 1-2.

[0081] Additionally, to confirm the binding of ICG with iron ions, the changes in the addition of iron ions at various concentrations to a certain amount of ICG were shown by way of images and absorption/fluorescence spectra. As shown in FIG. 2, it was confirmed that as the amount of iron ions increased, the hydrophobicity of ICG, which was well dispersed in water, increased to form a precipitate. Additionally, the absorption/fluorescence spectra showed that absorption decreased with the increase of the amount of iron ions and fluorescence was not shown, thus confirming that a hydrophobic composite was formed by the binding of ICG with iron ions. Additionally, it was confirmed that ICG and iron ions were present in the ICG-Fe composite at a binding ratio (molar ratio) of 3:1 using ICP-OES mass spectrometry and absorption spectra.

Example 1-2. Preparation of Nanoparticles Containing ICG-Fe Composite

[0082] 0.5 mg of the ICG-Fe composite prepared according to Example 1-1 was dissolved in 20 L of dimethyl sulfoxide (DMSO, Daejung Chemical & Metals Co., Ltd., Korea), and the resultant was added to 1 mL of an aqueous solution, in which Pluronic F127 (10 mg, Sigma-Aldrich, USA) as a polymeric surfactant was mixed, while dispersing by ultrasonic waves, and thereby ICG-Fe complex-encapsulated nanoparticles (ICG-Fe NPs) were prepared.

[0083] The schematic diagram of the design of the thus-prepared ICG-Fe NPs is shown in FIG. 1. The structure and morphology of the prepared nanoparticles were analyzed using transmission electron microscopy and absorption/fluorescence spectrum analysis, and the results are shown in FIG. 3. As shown in FIG. 3, the average diameter of the ICG-Fe NPs calculated from the TEM image was about 17 nm. The absorption/fluorescence spectrum of ICG was observed to confirm whether the ICG-Fe composite was encapsulated inside the prepared nanoparticles. The ICG absorption spectrum of the nanoparticles shifted to long wavelengths and fluorescence was hardly observed compared to the aqueous solution of ICG. From this result, it was confirmed that ICG was present inside of the nanoparticles.

Example 2: Evaluation of ICG-Fe NPs with Regard to Colloidal Stability and Photostability

Example 2-1. Evaluation of ICG-Fe NPs with Regard to Colloidal Stability in the Presence of Albumin

[0084] To confirm whether the ICG-Fe NPs prepared according to Example 1-2 can maintain colloidal stability even in the presence of albumin, which is a biomimetic environment, using the aqueous solution of ICG as the control group, the ICG-Fe NPs were each mixed with serum albumin (fatal bovine serum; FBS) at different concentrations, and the changes were observed on a 96-well plate using a fluorescence imaging device (IVIS). The results are shown in FIG. 4. As shown in FIG. 4, it was confirmed that the prepared ICG-Fe NPs were stably present while maintaining their structural form through the sustained fluorescence-quenching property, although the amount of serum albumin increased. In contrast, it was confirmed that ICG forms a dimer in an aquatic environment and partially exhibits a fluorescence-quenching property, and thus, ICG returned to a monomolecular state as it was exposed to high affinity proteins, thereby recovering fluorescence. These results indicate that the ICG bound to the iron ions is effectively encapsulated into the hydrophobic interior of the nanoparticles, and thereby the effect on the external ICG is reduced.

Example 2-2. Evaluation of Photostability of ICG-Fe NPs at the Time of Photoirradiation

[0085] To confirm whether the ICG-Fe NPs prepared according to Example 1-2 can maintain photostability at the time of photoirradiation, the changes in color and absorption/fluorescence signal intensity after irradiation with a laser (808 nm, 1 W) were examined, and the results are shown in FIG. 5. As shown in FIG. 5, it was confirmed that the prepared ICG-Fe NPs maintained their unique color even after laser irradiation for a certain period of time, compared to the aqueous solution of ICG and the ICG-albumin conjugate. Furthermore, based on the above result that the absorption/fluorescence signal intensity of the prepared ICG-Fe NPs was maintained even after laser irradiation at a level similar to that before laser irradiation, it was confirmed that the prepared ICG-Fe NPs have a greater improvement than the two control groups. These results are due to the fact that the nanoparticles were formed immediately as the iron ions were bound to ICG, and thereby the photodegradation by light was reduced.

Example 3: Evaluation of Photothermal and Photoacoustic Characteristics of ICG-Fe NPs

[0086] To confirm the photothermal efficiency of the ICG-Fe NPs prepared according to Example 1-2, the ICG-Fe NPs were irradiated with a laser (808 nm, 1 W) and the temperature of the solution was measured after irradiation. The results are shown in FIG. 6. As shown in FIG. 6, when the photothermal efficiencies were compared by having the aqueous solution of ICG and the ICG-albumin conjugate, which have similar absorbance, as control groups, it was confirmed that the prepared ICG-Fe NPs showed a temperature increase similar to those of the control groups at the beginning of laser irradiation, but the temperature of the prepared ICG-Fe NPs was maintained at about 60 C. under laser irradiation continued for 10 minutes. In contrast, it was confirmed that the two control groups showed a temperature increase to about 45 C. at the beginning of laser irradiation and then showed a phenomenon of temperature decrease due to photodegradation. Such an increase of photothermal efficiency of the ICG-Fe NPs is thought to be due to fluorescence of ICG, which competitively acts on the photothermal reaction and is present within the nanoparticles in a fluorescence-quenched state.

[0087] The increase in the photothermal efficiency may cause an improvement of the photoacoustic characteristic that occurs based on the photothermal effect. To confirm the photoacoustic characteristic of the prepared ICG-Fe NPs, the photoacoustic signal image of the nanoparticles obtained by the multispectral optoacoustic tomography (MSOT) device is shown in FIG. 7. As shown in FIG. 7, it was confirmed that ICG-Fe NPs show a photoacoustic signal.

Example 4: Evaluation of Toxicity of ICG-Fe NPs in Animal Models

[0088] 200 L of PBS and an aqueous solution of ICG, as control groups, and the ICG-Fe NPs prepared according to Example 1-2 were each intravenously injected to 5-week-old male nude mice (Orientbio Inc., Korea). Two days later, blood and liver/kidney tissues were collected and evaluated for toxicity. The blood liver levels and the tissue images observed by immunostaining are shown in FIG. 8. As shown in FIG. 8, the AST/ALT values for 3 groups of materials were shown to be at normal levels, and no abnormalities were found in immunostained liver/kidney tissues. These results indicate that the ICG-Fe NPs according to the present invention do not exhibit toxicity in a living environment.

Example 5: Evaluation of Characteristic of ICG-Fe NPs of Cancer Cell Accumulation in Cancer Disease Model and Photothermal Therapy

Example 5-1. Evaluation of Characteristic of ICG-Fe NPs of Cancer Cell Accumulation in Cancer Disease Model Via Fluorescence Imaging

[0089] The model with a cancer disease was prepared by subcutaneous injection of 110.sup.7 HT-29 cells (human colon cancer, Korean Cell Line Bank) on the left thigh of 5-week-old male nude mice (Orientbio Inc., Korea). After 2 weeks of cancer cell transplantation, solid tumors were confirmed to be formed, and then subsequent experiments were performed. Since the ICG-Fe NPs prepared according to Example 1-2 did not exhibit a fluorescence characteristic, 200 L of nanoparticles, which were prepared by introducing Cy5.5 (a different fluorescent material), were intravenously injected. Fluorescence imaging was performed before/after administration of the nanoparticles, and the results are shown in FIG. 9. As shown in FIG. 9, while the mouse injected with the ICG solution (control group) did not show fluorescence at the cancer site, the mouse injected with Cy5.5-labeled nanoparticles showed fluorescence due to the accumulation of the nanoparticles at the cancer site. These results indicate that ICG-Fe NPs according to the present invention have a characteristic of selective accumulation in cancer tissues.

Example 5-2. Evaluation of Photothermal Therapy Effect of ICG-Fe NPs in Cancer Disease Model

[0090] To mice model of cancer disease prepared according to Example 5-1, 200 L of ICG-Fe NPs prepared according to Example 2 and PBS were intravenously injected, and 1 and 6 hours thereafter, a partial laser treatment (808 nm, 2 W, 5 min) was performed on the cancer site. Material injections and laser treatments were repeated 3 times at 2 day intervals, and the results are shown in FIG. 10. As shown in FIG. 10, when 3 treatments were performed after the injection, the cancer model injected with PBS showed no therapeutic effect, whereas the mice injected with the nanoparticles showed a thrombosis in the cancer site due to the effect of hyperthermia treatment. These results indicate that the ICG-Fe NPs according to the present invention can be used for phototherapy of cancer.