Methylene blue nanoparticle for bioimaging and photodynamic therapy and use thereof

Abstract

The present invention relates to a methylene blue nanoparticle for bioimaging and photodynamic therapy, and a use thereof as a cancer therapeutic agent and a contrast agent. The methylene blue nanoparticle of the present invention for use as a topical cancer targeting photo therapeutic agent is composed of only a material of which the composition is clinically used or derived from human bodies, and thus a nanopreparation in which a barrier to clinical entry is low and the possibility of commercialization is very high, exhibits near-infrared fluorescence along with cancer targeting property, capacity of generating singlet oxygen and the like, and thus may be used for both bioimaging diagnosis such as optical imaging, and cancer targeting photodynamic therapy.

Claims

1. A methylene blue nanoparticle comprising a methylene blue-fatty acid complex and an amphiphilic copolymer of pluronic F-68 which comprises a polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer, wherein the methylene blue-fatty acid complex is enclosed in a micelle formed by the amphiphilic copolymer, and wherein the methylene blue nanoparticle has a diameter of 80 to 100 nm and is self-assembled in an aqueous environment.

2. The nanoparticle of claim 1, wherein the fatty acid is oleic acid or a salt thereof.

3. A composition for cancer photodynamic therapy, comprising the nanoparticle of claim 1.

4. A topical agent for cancer photodynamic therapy, comprising the nanoparticle of claim 1.

5. The composition of claim 3, wherein the cancer is breast cancer.

6. A contrast agent composition comprising the nanoparticle of claim 1.

7. The composition of claim 6, wherein the composition is for in vivo imaging diagnosis of cancer.

8. The composition of claim 6, wherein the composition is to be used as a contrast agent indicating a cancer resection margin in an image-guided operation.

9. The composition of claim 6, wherein the composition is to be used in cancer targeting photodynamic therapy and in vivo imaging diagnosis of cancer at the same time.

10. The methylene blue nanoparticle of claim 1, wherein the amphiphilic copolymer directly binds the methylene blue-fatty acid complex.

11. The methylene blue nanoparticle of claim 1, wherein the methylene blue nanoparticle consists of the methylene blue-fatty acid complex and the amphiphilic copolymer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments and together with the description serve to explain the principles of the disclosure.

(3) In the drawings:

(4) FIG. 1 is a schematic view of forming self-assembled nanoparticles including methylene blue as a photosensitizer in water.

(5) FIG. 2a shows the solubility of methylene blue; FIG. 2b is a graph showing absorbance of an aqueous solution of nanoparticles and methylene blue prepared in Example 1, which is measured by UV-visible spectrometer (Agilent 8453); and FIG. 2c is a graph showing fluorescence wavelength spectra of the aqueous solution, which is measured by fluorescence spectrophotometer (Hitachi F-7000, wavelength calibrated for excitation and emission).

(6) FIG. 3a is a graph showing the size of the nanoparticles prepared in Example 1, which is measured by dynamic light scattering; and FIG. 3b is a photograph of the nanoparticles observed by transmission electron microscope (TEM, CM30, FEI/Philips, 200 kV).

(7) FIG. 4 is a table in which the nanoparticles prepared in Example 1 are measured in terms of capacity of generating singlet oxygen in Example 2.

(8) FIGS. 5a, 5b, 5c, 5d, and 5e are fluorescence and optical photographs of each cancer cell in a control (CTRL) and after a methylene blue aqueous solution (MB Sol.) and nanoparticles (MB NPs) are injected, which are taken in Example 3 (1). (FIG. 5aMDA-MB-231; FIG. 5bMCF-7; FIG. 5cPC3; FIG. 5dHeLa; and FIG. 5eHT-29)

(9) FIGS. 6a, 6b, and 6c are fluorescence and optical photographs of each normal cell in a control (CTRL) and after a methylene blue aqueous solution (MB Sol.) and nanoparticles (MB NPs) are injected, which are taken in Example 3 (1). (FIG. 6aMRC-5; FIG. 6bClone 1-5c-4; and FIG. 6cNIH3T3)

(10) FIGS. 7a, 7b, 7c, 7d, 7d, and 7e are graphs of cytotoxicity (Dark) and photo-toxicity (Photo) of nanoparticles for each cancer cell, which are measured in Example 3 (2). (FIG. 7aMDA-MB-231 cell; FIG. 7bMCF-7 cell; FIG. 7cPC-3 cell; FIG. 7dHeLa cell; and FIG. 7eHT-29 cell)

(11) FIGS. 8a, 8b, and 8c are graphs of cytotoxicity (Dark) and phototoxicity (Photo) of nanoparticles for each normal cell, which are measured in Example 3 (2). (FIG. 8anormal cells MRC-5; FIG. 8bClone-1-5c-4; and FIG. 8cNIH3T3)

(12) FIG. 9 is photographs in which a change in shape of cell is observed over the time of irradiation of a light source on a HeLa cell into which MB NPs have been injected, which are taken in Example 3 (2).

(13) FIG. 10 is graphs in which the material absorption and apoptosis capacity of each cancer cell into which MB NPs have been injected in Example 3 (3) are measured by fluorescence activated cell sorter (FACS, EMD Millipore Corporation, USA).

(14) FIG. 11 is graphs in which the material absorption and apoptosis capacity of each normal cell into which MB NPs have been injected in Example 3 (3) are measured by fluorescence activated cell sorter (FACS, EMD Millipore Corporation, USA).

(15) FIG. 12 is photographs in which capacity of MB NPs and MB Sol. infiltrated into tissue is confirmed in Example 4 (1).

(16) FIG. 13a is a photograph in which image diagnosis behaviors of methylene blue nanoparticles (MB NPs) in an initial cancer model are confirmed by a topical cancer targeting contrast in Example 4 (2). FIG. 13b is a photograph showing image of methylene blue aqueous solution (MB Sol.) after topical administration.

(17) FIG. 14 is photographs in which the apoptosis capacity of MB NPs and the possibility of photodynamic therapy using an initial cancer model are confirmed in Example 4 (3).

(18) FIG. 15a shows photographs of a cancer site and FIG. 15b is a graph of change in size of cancer, of an animal model over time according to the photodynamic therapy, which are prepared in Example 4 (4).

(19) FIG. 16 is a graph in which the body weight of an animal (PDT) which has been subjected to photodynamic therapy is compared with the body weight of a control (Ctrl) which has not been subjected to photodynamic therapy in Example 4 (4).

DETAILED DESCRIPTION OF INVENTION

(20) Description will now be given in detail of the exemplary embodiments, with reference to the accompanying drawings. For the sake of brief description with reference to the drawings, the same or equivalent components will be provided with the same reference numbers, and description thereof will not be repeated.

(21) Hereinafter, the present invention will be described in more detail through the Examples. These Examples are provided only for more specifically describing the present invention, and it will be obvious to a person with ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these Examples.

EXAMPLE

Example 1

Formation of Self-Assembled Nanoparticles Comprising Methylene Blue As Photosensitizer in Aqueous Environment

(22) Preparation and Evaluation of Self-assembled Nanoparticles Using Oleate

(23) (1) Hydrophobic Modification of Methylene Blue/Sodium Oleate by Electrostatic Force

(24) 20 mg of methylene blue (MB, Aldrich Chemical Co.) and 30 mg of sodium oleate (O, Aldrich Chemical Co.) were dissolved in 100 mL of tetrahydrofuran (THF, Daejung Chemical Industry Co., Ltd.) by heating at 60 to 90 C. for 1 to 5 minutes.

(25) A stable methylene blue/sodium oleate complex (MBO) was formed by electrostatic force and hydrophobic interaction in a molecular structure, thereby exhibiting high solubility to the solvent.

(26) Remnant sodium oleate and other impurities were removed from the MBO solution using a syringe filter (5 m), and then MBO was obtained by following freeze-drying.

(27) (2) Preparation and Evaluation of Nanoparticle Comprising MBO and Amphiphilic Polymer

(28) 0.2 mg of MBO obtained in (1) and 20 mg of an amphiphilic polymer Pluronic F-68 (purchased from Aldrich Chemical Co.) were added into THF solvent and sufficiently mixed, and then the solvent was completely removed. The mixture from which the solvent had been completely removed was uniformly dispersed in 2 ml of water to prepare methylene blue nanoparticles (MB NPs).

(29) The absorbance and fluorescence of methylene blue dissolved in water (MB Sol.) and methylene blue nanoparticles dispersed in water (MB NPs) were measured, and the results were as shown in FIGS. 2b and 2c, respectively. It can be confirmed from FIGS. 2b and 2c that due to formation of nanoparticles, the absorption wavelength and fluorescent wavelength of MB NPs were shifted to a short wavelength region compared to MB dissolved in water.

(30) The size of the nanoparticles was measured by Zetasisernano ZS (Malvern Instruments, UK) and the shape of the nanoparticles was observed by transmission electron microscope (TEM, CM30, PEI/Philips, 200 kV). The results were represented in FIGS. 3a and 3b, respectively. As can be confirmed from FIGS. 3a and 3b, the nanoparticle was observed to have a spherical form with a diameter of 80 to 100 nm.

(31) Preparation of Self-assembled Nanoparticles Using Stearate

(32) (1) Hydrophobic Modification of Methylene Blue/Sodium Stearate by Electrostatic Force

(33) As a comparative example, a mixture of methylene blue and sodium stearate (MBSt) was prepared in the same way as mentioned above, except using 30 mg of sodium stearate (St, purchased from Aldrich Chemical Co.) instead of sodium oleate.

(34) Although the combination of methylene blue and sodium oleate was completely dissolved in the solvent, the combination of a methylene blue and sodium stearate showed very low solubility to the solvent because sodium stearate was not dissolved in the solvent and thereby failed to modify methylene blue (see FIG. 2a).

(35) (2) Preparation of Nanoparticle Comprising MBSt and Amphiphilic Polymer

(36) An experiment was carried out to prepare methylene blue nanoparticles under the same condition using MBSt and 20 mg of an amphiphilic polymer Pluronic F-68 (Aldrich) in THF solvent. However, no stable methylene blue nanoparticle was obtained in the solvent because MBSt itself failed to form a stable complex in the solvent.

(37) It was confirmed from Example 1 that methylene blue can be efficiently enclosed within amphiphilic polymers in aqueous environment by electrostatically neutralizing and hydrophobically modifying methylene blue using fatty acid. The nanoparticles in which methylene blue is enclosed within amphiphilic polymers were found to have excellent structure stability maintaining their particle state through the size measurement and shape observation.

Example 2

Evaluation of Capacity of Generating Singlet Oxygen of Self-Assembled Nanoparticles (MB NPs) Comprising Methylene Blue As Photosensitizer in Aqueous Environment

(38) In order to use MB NPs dispersed in water as a photosensitizer for photodynamic therapy, the capacity of generating singlet oxygen of the nanoparticles according to the laser irradiation was determined and compared to that of MB dissolved in water (MB Sol.). A laser with a wavelength of 655 nm which is known to be capable of producing singlet oxygen (Chanchun New industries Optoelectronics Tech. Co., Ltd., ex=655 nm, 200 mW output power) was used.

(39) The amount of singlet oxygen produced was measured by a chemical method using N,N-Dimethyl-4-nitrosoaniline (Aldrich) which is combined with singlet oxygen to lose the inherent OD max value.

(40) FIG. 4 illustrates the result, confirming that MB NPs showed a capacity of generating singlet oxygen, which is equivalent to that of MB Sol. at 37 C. which is a temperature suitable for living organisms.

(41) By confirming the capacity of generating singlet oxygen at 37 C., the possibility of photodynamic therapy of methylene blue as a photosensitizer in vivo was confirmed.

Example 3

Evaluation of Cancer Cell Accumulation and Characteristics of Self-Assembled Nanoparticles Comprising Methylene Blue As Photosensitizer in Aqueous Environment

(42) (1) Evaluation of Accumulation of MB NPs in Cancer Cell

(43) In order to determine if the nanoparticles (MB NPs) prepared in Example 1 would be accumulated in the cancer cell, 110.sup.5 of each of cancer cells MDA-MB-231 (breast, mammary gland/human, Korean Cell Line Bank), MCF-7 (breast, mammary gland/human, Korean Cell Line Bank), PC-3 (prostate; grade 4; metastasis to bone/human, Korean Cell Line Bank), HeLa (cervix/human, Korean Cell Line Bank), and HT-29 (Colon/human, Korean Cell Line Bank) was respectively dispersed in 2 mL of cell culture solution [DMEM (WELGENE) culture solution was used for the HeLa cell, and RPMI1640 (WELGENE) culture solution was used for the other cells]. And then the solution was put into a dish for cell culture (35 mm, glass bottomed dish), and cultured in a culture chamber (5% CO.sub.2, 37 C.). After 24 hours, the dish was washed with 2 mL of DPBS (WELGENE), and 1.8 mL of the culture solution and 0.2 mL of the nanoparticles were added thereto, and the resulting dish was stored in a culture chamber (5% CO.sub.2, 37 C.) for 1 hour. The stored cell culture dish was washed with 2 mL of DPBS, cells were fixed with 1 mL of a cell fixation solution, and then fluorescence images were observed by fluorescence microscope (LEICA DMI3000B equipped with a Nuance FX multispectral imaging system, CRI).

(44) For a comparative experiment, the same experiment was carried out by using a solution (MB Sol.) obtained by dissolving the same amount of MB used in the aforementioned experiment in water. The experimental results for MB NPs and MB Sol. are illustrated in FIGS. 5a to 5e.

(45) Further, in order to check if MB NPs would be accumulated in normal cells other than the cancer cells, experiment was carried out under the same condition regarding MRC-5 (lung/human, Korean Cell Line Bank), clone 1-5c-4(conjunctiva/human, Korean Cell Line Bank), and NIH/3T3(embryo/mouse, Korean Cell Line Bank) cells, and the results are shown in FIGS. 6a to 6c.

(46) As a result of the experiment, the MB Sol. had so low infiltration capacity into cell that MB Sol. was not found in the cell, whereas the MB NPs which formed nanoparticles in aqueous environment due to hydrophobic modification of methylene blue using a fatty acid salt and an amphiphilic polymer were found to have improved infiltration capacity and nanoparticle stability in the cell ambient environment.

(47) (2) Evaluation of Cancer Cell Photo-Toxicity of MB NPs

(48) In order to confirm of the cytotoxicity of MB NPs, 200 L of a cell culture solution in which 110.sup.4 ea of cells were dispersed was put into a dish for cell culture (96-well plate) and cultured (5% CO.sub.2, 37 C.) in an artificial culture chamber for 24 hours, and the dish was washed with DPBS, and then a mixture solution of 20 L of MB NPs and 180 L of a cell culture solution was added to the cell, and the resulting culture solution was cultured in the same artificial culture chamber for 1 hour. After the dish was washed with DPBS, the number of each cell was observed by the MTT analysis method (Document 7: T. Mosmann et al., Journal of Immunological Method 65:55-63 (1983)).

(49) Further, in order to evaluate the photo-toxicity of MB NPs for each cancer cell, experiment was performed in the same manner as mentioned above, and the number of cells by photo-toxicity was determined by irradiating laser onto each cell for 20 minutes, and then performing an MTT analysis.

(50) The experiment was performed on cancer cells and normal cells, and MDA-MB-231 cell, MCF-7 cell, PC-3 cell, HeLa cell, and HT-29 cell are illustrated in FIGS. 7a, 7b, 7c, 7d, and 7e, respectively, and normal cells MRC-5, Clone-1-5c-4, and NIH3T3 are illustrated in FIGS. 8a to 8c, respectively.

(51) The experiment of evaluating cytotoxicity and photo-toxicity by MB Sol. was also performed in the same manner as described above, but cytotoxicity and photo-toxicity for a control were also almost the same as described above, so that it was confirmed that there are no cytotoxicity of the material itself, nor cytotoxicity by a light source.

(52) In order to observe the change in cell form over time due to the toxicity by a light source, a culture solution in which 110.sup.5 ea of cells were dispersed was put into a dish for culture (35 mm), and after 24 hours, the dish was washed with 2 mL of DPBS to inject 1.8 mL of the culture solution and 0.2 mL of the prepared MB NPs into the dish. Then, after 1 hour, the sample was observed by a microscope (LEICA DMI3000B equipped with a Nuance FX multispectral imaging system, CRI) along with irradiation of a light source, and the result is shown in FIG. 9.

(53) Through the experiment, it was found that even though nanoparticles with improved infiltration capacity were absorbed in the cell, the cytotoxicity of the material was not significant for each cell, whereas for the cancer cell in which the nanoparticles were absorbed, photo-toxicity by a light source was mostly observed, and it was found that specifically for the breast cancer cells (MDA-MB-231 and MCF-7), the effect was still excellent. As described above, the possibility of cancer cell targeting therapy was confirmed.

(54) (3) Evaluation of Apoptosis-Inducing Capacity of MB NPs Accumulated in Cancer Cell by Laser

(55) In order to evaluate the apoptosis-inducing capacity of MB NPs by a light source, 2 mL of a cell culture solution in which 210.sup.5 ea of cells were dispersed was put into a dish for cell culture (12-well plate) and cultured (5% CO.sub.2, 37 C.) in an artificial culture chamber for 48 hours. After washing with 2 mL of DPBS, a mixture solution of 1.8 mL of the culture solution and 0.2 mL was injected into the dish for cell culture, and then cultured in an artificial culture chamber for 1 hour to absorb the material. Then, after washing with DPBS, irradiation was performed by a light source using laser with a wavelength of 655 nm for 5 minutes, DPBS was removed, and then a mixture solution Annexin V/FITC of 0.5 mL of a binding buffer and 10 L of FITC was injected into the dish, and the resulting dish was stored in an artificial culture chamber for 5 minutes. After washed with DPBS, the dish was treated with trypsin EDTA (WELGENE) to separate cells from the dish for cell culture. The separated cells were dispersed in 1 mL of DPBS to perform measurement by a fluorescence activated cell sorter (FACS, guava easyCyteSingle Sample Flow Cytometer, EMD Millipore Corporation, USA) device.

(56) MB NPs were absorbed in Example 3 (3), an effective apoptosis-inducing capacity could be confirmed from most of the cancer cells through the measurement of the apoptosis-inducing capacity by Annexin V/FITC from the cancer cell irradiated by laser, and it was confirmed that this was a result having the same aspect when compared to the cell photo-toxicity in Example 3 (2). Furthermore, it was confirmed that specifically for the breast cancer cells (MDA-MB-231 and MCF-7) among the cancer cells, the apoptosis-inducing capacity and the photo-toxicity in Example 3 (2) were excellent, and through this, the possibility of a specific cancer cell targeted therapy could be confirmed. The results in Example 3 (3) were illustrated in FIGS. 10 and 11, and from this, the apoptosis-inducing capacities of the cancer cells and the normal cells could be respectively confirmed.

Example 4

Evaluation of Cancer Targeting Accumulation and Characteristics of MB NPs after Being Topically Injected into Living Organism

(57) (1) Evaluation of Tissue Infiltration Capacity of MP NPs and MB Sol.

(58) Commercially available chicken breast meat (Moguchon Co., Ltd.) was cut into a predetermined size (3 cm3 cm3 cm), 0.5 mL of each of the MB NPs and MB Sol. prepared in Example 1 was applied onto the meat, and then the resulting sample was stored at 37 C. in a water bath. Thereafter, the sample was sufficiently washed with DPBS, and a fluorescence image was obtained by a fluorescence image device 12-bit CCD camera (Kodak Image Station 4000 MM, ex: 625 nm/em: 700 nm). The result is illustrated in FIG. 12.

(59) Since methylene blue is present in the form of nanoparticles embedded in the amphiphilic polymer, it was confirmed that tissue infiltration capacity had been significantly improved compared to methylene blue alone.

(60) (2) Evaluation of MB NPs for Topical Cancer Targeting in Imaging Diagnosis Using Cancer Model

(61) 60 L of a culture solution in which 110.sup.7 ea of the MDA-MB-231 cells were dispersed was injected into the left hip muscular site of a female rat (Balb/c-nu, 5.5 week old, Orient Bio Inc.). Thereafter, the growth of the cancer tissue for 4 to 5 weeks was confirmed by the naked eye, 60 L of nanoparticles (MB NPs) in which methylene blue was enclosed were topically injected into the cancer tissue site, and it was confirmed that the cancer tissue target had been accumulated by using a fluorescence image device (IVIS-Spectrum, Perkin-Elmer, USA). For a comparative experiment of the aforementioned experiment, an experiment using a methylene blue aqueous solution (MB Sol.) was carried out on an animal model prepared in the same manner as mentioned above and the results are as illustrated in FIGS. 13a and 13b.

(62) From the experiment, the cancer tissue targeting accumulation capacity of MB NPs was confirmed in the animal cancer model as well, and thereby the possibility of cancer targeting of MB NPs for in vivo imaging diagnosis of cancer was confirmed.

(63) (3) Evaluation of Accumulation and Photo Therapy Characteristics of MB NPs Using Initial Cancer Model

(64) In order to perform an accumulation test for nanoparticles in an initial cancer model, 110.sup.6 ea of SCC7 (Squamous Cell Carcinoma) cells dispersed in 60 L of the cell culture solution were subcutaneously injected into each of the left and right hip muscular sites of a male rat (Balb/c-nu, 5.5 week old, Orient Bio Inc.). After 2 hours, 60 L of MB NPs were subcutaneously injected into the left site where the cancer cell had been injected, and after 2 hours, the material was sufficiently absorbed into the cell, and then laser was irradiated onto the left hip site for 20 minutes. Thereafter, 60 L of Annexin V/FITC (a mixture solution of 100 L of a binding buffer and 10 L of FITC) was subcutaneously injected into the left and right initial cancer sites to evaluate the apoptosis capacity, and a fluorescence image was obtained from the initial cancer model using a fluorescence image device (IVIS-Spectrum, Perkin-Elmer, USA) to evaluate the apoptosis capacity of MB NPs by photo-toxicity. The results are illustrated in FIG. 14.

(65) From the experiment, it was confirmed that MB NPs in which hydrophobically modified methylene blue is introduced into the amphiphilic polymer as a photosensitizer could be used to cause apoptosis, and thereby in vivo photodynamic therapy was feasible.

(66) (4) Evaluation of Photodynamic Therapy Efficacy and Biotoxicity of In Vivo MB NPs

(67) In order to evaluate an in vivo photodynamic therapy efficacy, 60 L of a culture solution in which 110.sup.7 ea of the MDA-MB-231 cell expected to have an excellent photodynamic therapy effect by MB NPs as confirmed in Example 3 (2) was dispersed was injected into the left hip muscular site of a female rat (Balb/c-nu, 5.5 week old, Orient Bio Inc.), the growth of the cancer tissue for 4 to 5 weeks was confirmed by the naked eye, and then 60 L of MB NPs were subcutaneously injected into the cancer tissue site. Thereafter, the photodynamic therapy (irradiation of laser with a wavelength of 655 nm for 10 minutes) was performed 8 times in total at the interval of 2 to 3 days, the result observed for 28 days is illustrated in FIG. 15a, and the change in size of cancer (=short axis.sup.2 X long axis) over passage of time is illustrated in FIG. 15b.

(68) Furthermore, in order to evaluate the in vivo toxicity of MB NPs, a graph, which compares the weights of a control (Ctrl) which was not subjected to photodynamic therapy and an experimental group (PDT) which was subjected to photodynamic therapy, is illustrated in FIG. 16.

(69) From the experiment, an excellent therapy effect was confirmed during the photodynamic therapy using MB NPs as a photosensitizer, and it could be confirmed that the in vivo toxicity of MB NPs by a cancer ambient topical injection was not shown.

(70) The foregoing embodiments and advantages are merely exemplary and are not to be considered as limiting the present invention. The present teachings can be readily applied to other types of apparatuses. This description is intended to be illustrative, and not to limit the scope of the claims. Many alternatives, modifications, and variations will be apparent to those skilled in the art. The features, structures, methods, and other characteristics of the exemplary embodiments described herein may be combined in various ways to obtain additional and/or alternative exemplary embodiments.

(71) As the present features may be embodied in several forms without departing from the characteristics thereof, it should also be understood that the above-described embodiments are not limited by any of the details of the foregoing description, unless otherwise specified, but rather should be considered broadly within its scope as defined in the appended claims, and therefore all changes and modifications that fall within the metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the appended claims.