Methylene blue complex for treating skin disease and its use thereof

Abstract

The present invention relates to complex particles using methylene blue for treating a skin disease caused by Propionibacterium acnes or Staphylococcus aureus and a composition for treatment including the complex particles. The complex particles in the present invention can be used as a photosensitizer for a photodynamic therapy and complex particles having a micelle form in which hydrophilic methylene blue and two hydrophobic organic acids are combined, and as a result, pore penetration is easy and an occlusion time can be significantly reduced to 30 minutes as compared with conventional phototherapy requiring an occlusion time of 1 hour to 3 hours. Further, in order to reduce side effects of a residual photosensitizer in phototherapy using an existing photosensitizer due to photoreaction and photobleaching of the methylene blue-organic acid complex, a light protection (light blocking or light shielding) time when contact of light needs to be avoided for 24 hours or more after treatment can be significantly reduced to 3 hours, and target treatment for Propionibacterium acnes, Staphylococcus aureus, or the like which is a cause of acne is possible.

Claims

1. A methylene blue complex for phototherapy of a skin disease, comprising a methylene blue, a first organic acid, and a second organic acid, wherein the complex has a micelle form in which a mixture of the methylene blue and the first organic acid is positioned at a center of the complex and the second organic acid is positioned at an outside of the mixture, the first organic acid is selected from the group of docosahexaenoic acid (DHA), indole-3-acetic acid (IAA), tranexamic acid, salicylic acid, linoleic acid, linolenic acid, salicylsalicylic acid, acetylsalicylic acid, and methyl salicylic acid, the second organic acid is oleic acid, the skin disease is a skin disease caused by Staphylococcus aureus or Propionibacterium acnes, and wherein the methylene blue, the first organic acid, and the second organic acid forming the complex micelle have mass ratios of: a mass of the first organic acid is 1 to 5 times greater than the mass of the methylene blue and a mass of the second organic acid is 50 to 500 times greater than the mass of the mixture of the methylene blue and the first organic acid.

2. The methylene blue complex for phototherapy of a skin disease of claim 1, wherein in the phototherapy of the skin disease using the complex, an occlusion time after coating the complex is 1 hour or less, and a light protection time after treatment is 3 hours or less.

3. The methylene blue complex for phototherapy of a skin disease of claim 2, wherein a diameter of the complex is 50 nm or more and 100 μm or less.

4. A composition effective for treating acne caused by acne bacteria or Staphylococcus aureus, consisting essentially of a methylene blue complex composed of a methylene blue, a first organic acid, and a second organic acid, wherein the complex has a micelle form in which a mixture of the methylene blue and the first organic acid is positioned at a center of the complex and the second organic acid is positioned at an outside of the mixture, wherein the first organic acid is selected from the group of docosahexaenoic acid (DHA), indole-3-acetic acid (IAA), tranexamic acid, salicylic acid, linoleic acid, linolenic acid, salicylsalicylic acid, acetylsalicylic acid, and methyl salicylic acid, the second organic acid is oleic acid, and wherein the methylene blue, the first organic acid, and the second organic acid forming the complex micelle have mass ratios of: a mass of the first organic acid is 1 to 5 times greater than the mass of the methylene blue and a mass of the second organic acid is 50 to 500 times greater than the mass of the mixture of the methylene blue and the first organic acid.

5. The composition for phototherapy of a skin disease of claim 4, wherein in the phototherapy of the skin disease using the complex, an occlusion time after coating the nanoparticles is 1 hour or less, and a light protection time after treatment is 3 hours or less.

6. The composition for phototherapy of a skin disease of claim 5, wherein a diameter of the complex is 50 nm or more and 100 μm or less.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic diagram of a complex particle composed of methylene blue-salicylic acid-oleic acid of the present invention.

(2) FIG. 2 is a graph illustrated by measuring absorbance and fluorescence intensity when micelle-type complex particles composed of methylene blue and organic acids are dispersed in water.

(3) FIGS. 3A and 3B are a table and a distribution diagram illustrated by measuring a size of the complex particle by using a Zetasiser nano ZS and a transmission electron microscope in nanoparticles prepared in Example 1, respectively.

(4) FIG. 4 is a graph illustrating productivity of singlet oxygen when complex particles MBX NPs formed of methylene blue and organic acids of the present invention in which the methylene blue is encapsulated in the complex particle is compared with a methylene blue solution MB Sol. in which the same amount of methylene blue is dissolved in water.

(5) FIG. 5 is a graph illustrating evaluation of photo-toxicity and dark-toxicity in Example 3.

(6) FIG. 6A and FIG. 6B illustrate evaluation of photo-toxicity of complex particle formed by using methylene blue and salicylic acid in Example 5, in which FIG. 6A illustrates an image of Propionibacterium acnes (P. Acnes) and FIG. 6B illustrates evaluation of photo-toxicity for P. Acnes.

MODES OF THE INVENTION

(7) Hereinafter, the present invention will be described in more detail through Examples. These Examples are just to describe the present invention in more detail and it is apparent to those skilled in the art that the scope of the present invention is not limited to these Examples.

EXAMPLES

Example 1: Preparation of Hydrophobic Complex of Photosensitizer (Methylene Blue) and Organic Acids

(8) (1) Hydrophobicization of Methylene Blue/DHA by Electrostatic Force

(9) 20 mg of methylene blue (MB, purchased from Aldrich) and 20 mg of DHA (D, purchased from Aldrich) were heated in 50 mL of tetrahydrofuran (THF, purchased from Daejeong Chemical) for 1 to 5 minutes at 60 to 90° C. and the MB was dissolved. The mixture in which the MB was dissolved was filtered by using a hydrophobic injection filter (0.2 μm), and the obtained filtrate was dried. The MB complex dissolved in the organic layer was extracted by using chloroform and water, purified and lyophilized to obtain an MBD (a complex of the MB and the DHA).

(10) (2) Hydrophobicization of Methylene Blue/IAA by Electrostatic Force

(11) 20 mg of methylene blue (MB, purchased from Aldrich) and 20 mg of indole-3-acetic acid (I, purchased from Aldrich) were heated in 50 mL of tetrahydrofuran (THF, purchased from Daejeong Chemical) for 1 to 5 minutes at 60 to 90° C. and the MB was dissolved. The mixture in which the MB was dissolved was filtered by using a hydrophobic injection filter (0.2 μm), and the obtained filtrate was dried. The MB complex dissolved in the organic layer was extracted by using chloroform and water, purified and lyophilized to obtain a MBI (a complex of the MB and the IAA).

(12) (3) Hydrophobicization of Methylene Blue/Tranexamic Acid by Electrostatic Force

(13) 20 mg of methylene blue (MB, purchased from Aldrich) and 20 mg of tranexamic acid (T, purchased from Aldrich) were heated in 50 mL of tetrahydrofuran (THF, purchased from Daejeong Chemical) for 1 to 5 minutes at 60 to 90° C. and the MB was dissolved. The mixture in which the MB was dissolved was filtered by using a hydrophobic injection filter (0.2 μm), and the obtained filtrate was dried. The MB complex dissolved in the organic layer was extracted by using chloroform and water, purified and lyophilized to obtain a MBT (a complex of the MB and the tranexamic acid).

(14) (4) Hydrophobicization of Methylene Blue/Salicylic Acid Salt by Electrostatic Force

(15) 20 mg of methylene blue (MB, purchased from Aldrich) and 20 mg of salicylic acid salt (S, purchased from Aldrich) were heated in 50 mL of tetrahydrofuran (THF, purchased from Daejeong Chemical) for 1 to 5 minutes at 60 to 90° C. and the MB was dissolved. The mixture in which the MB was dissolved was filtered by using a hydrophobic injection filter (0.2 μm), and the obtained filtrate was dried. The MB complex dissolved in the organic layer was extracted by using chloroform and water, purified and lyophilized to obtain a MBS (a complex of the MB and the salicylic acid).

(16) (5) Hydrophobicization of Methylene Blue/Ascorbic Acid by Electrostatic Force

(17) 20 mg of methylene blue (MB, purchased from Aldrich) and 20 mg of ascorbic acid (A, purchased from Aldrich) were heated in 50 mL of tetrahydrofuran (THF, purchased from Daejeong Chemical) for 1 to 5 minutes at 60 to 90° C. and the MB was dissolved. The mixture in which the MB was dissolved was filtered by using a hydrophobic injection filter (0.2 μm), and the obtained filtrate was dried. The MB complex dissolved in the organic layer was extracted by using chloroform and water, purified and lyophilized to obtain a MBA (a complex of the MB and the ascorbic acid).

Example 2: Formation of Self-Assembled Complex Particle Using Complex of Photosensitizer (Methylene Blue) and Organic Acid Hydrophobicized in Aqueous Environment

(18) (1) Preparation of Nanoparticles Containing MBD and Amphiphilic Material and Evaluation Thereof

(19) 0.2 mg of the MBD obtained in the Example 1 (1) was sufficiently dispersed by using 2 mL of an aqueous solution in which 20 mg of oleate (purchased from Aldrich) was dissolved to prepare complex particles MBD NPs.

(20) From FIG. 2 which is a graph illustrated by measuring absorbance and fluorescence of a MB (MB Sol.) dissolved in water and the complex particles MBD NPs dispersed in the water, it was confirmed that an absorption wavelength and a fluorescence wavelength of the MB dissolved in the water moved to a short wavelength region by forming the complex particles.

(21) Further, the size and the shape of the complex particle were measured by using a Zetasiser nano ZS (Malvern Instruments, UK) and observed by a transmission electron microscope (TEM, Tecnai) and then illustrated in FIGS. 3A and 3B, respectively. From FIGS. 3A and 3B, it was confirmed that the complex particles were spherical particles having a diameter of 50 to 200 nm.

(22) (2) Preparation of Complex Particles Containing MBI and Amphiphilic Material and Evaluation Thereof

(23) 0.2 mg of the MBI obtained in the Example 1 (2) was sufficiently dispersed by using 2 mL of an aqueous solution in which 20 mg of oleate (purchased from Aldrich) was dissolved to prepare complex particles MBD NPs.

(24) From FIG. 2 which is a graph illustrated by measuring absorbance and fluorescence of a MB (MB Sol.) dissolved in water and the complex particles MBI NPs dispersed in the water, it was confirmed that an absorption wavelength and a fluorescence wavelength of the MB dissolved in the water moved to a short wavelength region by forming the complex particles.

(25) Further, the size and the shape of the complex particle were measured by using a Zetasiser nano ZS (Malvern Instruments, UK) and observed by a transmission electron microscope (TEM, Tecnai) and then illustrated in FIGS. 3A and 3B, respectively. From FIGS. 3A and 3B, it was confirmed that the complex particles were spherical particles having a diameter of 50 to 200 nm.

(26) (3) Preparation of Complex Particles Containing MBT and Amphiphilic Material and Evaluation Thereof

(27) 0.2 mg of the MBT obtained in the Example 1 (3) was sufficiently dispersed by using 2 mL of an aqueous solution in which 20 mg of oleate (purchased from Aldrich) to prepare complex particles MBT NPs.

(28) From FIG. 2 which is a graph illustrated by measuring absorbance and fluorescence of a MB (MB Sol.) dissolved in water and the complex particles MBT NPs dispersed in the water, it was confirmed that an absorption wavelength and a fluorescence wavelength of the MB dissolved in the water moved to a short wavelength region by forming the complex particles.

(29) Further, the size and the shape of the complex particle were measured by using a Zetasiser nano ZS (Malvern Instruments, UK) and observed by a transmission electron microscope (TEM, Tecnai) and then illustrated in FIGS. 3A and 3B, respectively. From FIGS. 3A and 3B, it was confirmed that the complex particles were spherical particles having a diameter of 50 to 200 nm.

(30) (4) Preparation of Complex Particles Containing MBS and Amphiphilic Material and Evaluation Thereof

(31) 0.2 mg of the MBS obtained in the Example 1 (4) was sufficiently dispersed by using 2 mL of an aqueous solution in which 20 mg of oleate (purchased from Aldrich) to prepare complex particles MBS NPs.

(32) From FIG. 2 which is a graph illustrated by measuring absorbance and fluorescence of a MB (MB Sol.) dissolved in water and the complex particles MBS NPs dispersed in the water, it was confirmed that an absorption wavelength and a fluorescence wavelength of the MB dissolved in the water moved to a short wavelength region by forming the complex particles.

(33) Further, the size and the shape of the complex particle were measured by using a Zetasiser nano ZS (Malvern Instruments, UK) and observed by a transmission electron microscope (TEM, Tecnai) and then illustrated in FIGS. 3A and 3B, respectively. From FIGS. 3A and 3B, it was confirmed that the complex particles were spherical particles having a diameter of 50 to 200 nm.

(34) (5) Preparation of Complex Particles Containing MBA and Amphiphilic Material and Evaluation Thereof

(35) 0.2 mg of the MBA obtained in the Example 1 (5) was sufficiently dispersed by using 2 mL of an aqueous solution in which 20 mg of oleate (purchased from Aldrich) to prepare complex particles MBA NPs.

(36) From FIG. 2 which is a graph illustrated by measuring absorbance and fluorescence of a MB (MB Sol.) dissolved in water and the complex particles MBA NPs dispersed in the water, it was confirmed that an absorption wavelength and a fluorescence wavelength of the MB dissolved in the water moved to a short wavelength region by forming the complex particles.

(37) Further, the size and the shape of the complex particle were measured by using a Zetasiser nano ZS (Malvern Instruments, UK) and observed by a transmission electron microscope (TEM, Tecnai) and then illustrated in FIGS. 3A and 3B, respectively. From FIGS. 3A and 3B, it was confirmed that the complex particles were spherical particles having a diameter of 50 to 200 nm.

(38) In the Example 1, electrostatic neutralization with the MB and hydrophobicization by the organic acid were performed, and in the Example 2 using them, the MB can be efficiently encapsulated in oleic acid which was fatty acid in an aqueous environment. In the aqueous environment, the complex particles in which the MB was encapsulated in the amphiphilic material had excellent stability to maintain the particle state and the stability was confirmed through the size and the shape.

Example 3: Evaluation of Productivity of Singlet Oxygen of Self-Assembled Complex Particles Containing Methylene Blue as Photosensitizer in Aqueous Environment

(39) In order to use the MBS NPs dispersed in the water as a photosensitizer for photodynamic therapy, productivity of singlet oxygen of nanoparticles according to irradiation of a light source was confirmed by comparing with the MB (MB Sol.). The used light source was use a light source (Healite II 633, Lutronics, Co., Ltd.) having a wavelength of 633 nm, in which the MB was known to generate the singlet oxygen.

(40) A generated amount of singlet oxygen may be measured by a chemical method using N,N-dimethyl-4-nitrosoaniline (purchased from Aldrich) which was coupled with the singlet oxygen to lose a unique OD max value.

(41) FIG. 4 illustrates the result and confirmed productivity of the singlet oxygen which had no difference when the MBD, MBA, MBT, MBS, and MBA NPs in which the MB was encapsulated in the complex particle were compared with the MB Sol. in which the same amount of MB was dissolved in the water.

Example 4: Evaluation of Photo-Toxicity and Dark-Toxicity for Staphylococcus Aureus of Self-Assembled Complex Particles Containing Methylene Blue as Photosensitizer in Aqueous Environment

(42) (1) Evaluation of Photo-Toxicity Using Staphylococcus Aureus

(43) {circle around (1)} Evaluation of Photo-Toxicity of MBD NPs

(44) In order to evaluate photo-toxicity to bacteria of the complex particles MBD NPs prepared through the Examples 1 and 2 depending on a concentration, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Staphylococcus aureus (S. aureus) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of nanoparticles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 8.08 mg/mL, 4.04 mg/mL, and 0 mg/mL). For evaluation of photo-toxicity after 10 minutes, light (intensity 4) was irradiated for 10 minutes by using a Healite II 633 (purchased from Lutronics. Co., Ltd.). The bacteria per concentration after light irradiation were diluted to 1×10.sup.−6 and 1 mL was inoculated in a perifilm (purchased from 3M) and cultured in an incubator (37° C.) for 24 hours, and then the bacterial count was measured.

(45) As a control experiment, an MB aqueous solution corresponding to each concentration of the complex particles was prepared and the bacterial count was measured through the same process as the Example 4 (1) {circle around (1)}.

(46) Further, in order to confirm bacterial toxicity of only the complex particles except for the light source, evaluation of dark-toxicity was performed and illustrated in the Example 4 (2).

(47) {circle around (2)} Evaluation of Photo-Toxicity of MBI NPs

(48) In order to evaluate photo-toxicity to bacteria of the complex particles MBI NPs prepared through the Examples 1 and 2 depending on a concentration, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Staphylococcus aureus (S. aureus) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of nanoparticles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 8.08 mg/mL, 4.04 mg/mL, and 0 mg/mL). For evaluation of photo-toxicity after 10 minutes, light (intensity 4) was irradiated for 10 minutes by using a Healite II 633 (purchased from Lutronics. Co., Ltd.). The bacteria per concentration after light irradiation were diluted to 1×10.sup.−6 and 1 mL was inoculated in a perifilm (purchased from 3M) and cultured in an incubator (37° C.) for 24 hours, and then the bacterial count was measured.

(49) As a control experiment, an MB aqueous solution corresponding to each concentration of the complex particles was prepared and the bacterial count was measured through the same process as the Example 4 (1) {circle around (2)}.

(50) Further, in order to confirm bacterial toxicity of only the complex particles except for the light source, evaluation of dark-toxicity was performed and illustrated in the Example 4 (2).

(51) {circle around (3)} Evaluation of Photo-Toxicity of MBT NPs

(52) In order to evaluate photo-toxicity to bacteria of the complex particles MBT NPs prepared through the Examples 1 and 2 depending on a concentration, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Staphylococcus aureus (S. aureus) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of complex particles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 8.08 mg/mL, 4.04 mg/mL, and 0 mg/mL). For evaluation of photo-toxicity after 10 minutes, light (intensity 4) was irradiated for 10 minutes by using a Healite II 633 (purchased from Lutronics. Co., Ltd.). The bacteria per concentration after light irradiation were diluted to 1×10.sup.−6 and 1 mL was inoculated in a perifilm (purchased from 3M) and cultured in an incubator (37° C.) for 24 hours, and then the bacterial count was measured.

(53) As a control experiment, an MB aqueous solution corresponding to each concentration of the complex particles was prepared and the bacterial count was measured through the same process as the Example 4 (1) {circle around (3)}.

(54) Further, in order to confirm bacterial toxicity of only the complex particles except for the light source, evaluation of dark-toxicity was performed and illustrated in the Example 4 (2).

(55) {circle around (4)} Evaluation of Photo-Toxicity of MBS NPs

(56) In order to evaluate photo-toxicity to bacteria of the complex particles MBS NPs prepared through the Examples 1 and 2 depending on a concentration, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Staphylococcus aureus (S. aureus) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of complex particles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 4.04 mg/mL, and 0 mg/mL). For evaluation of photo-toxicity after 10 minutes, light (intensity 4) was irradiated for 10 minutes by using a Healite II 633 (purchased from Lutronics. Co., Ltd.). The bacteria per concentration after light irradiation were diluted to 1×10.sup.−6 and 1 mL was inoculated in a perifilm (purchased from 3M) and cultured in an incubator (37° C.) for 24 hours, and then the bacterial count was measured.

(57) As a control experiment, an MB aqueous solution corresponding to each concentration of the complex particles was prepared and the bacterial count was measured through the same process as the Example 4 (1) {circle around (4)}.

(58) Further, in order to confirm bacterial toxicity of only the complex particles except for the light source, evaluation of dark-toxicity was performed and illustrated in the Example 4 (2).

(59) {circle around (5)} Evaluation of Photo-Toxicity of MBA NPs

(60) In order to evaluate photo-toxicity to bacteria of the complex particles MBA NPs prepared through the Examples 1 and 2 depending on a concentration, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Staphylococcus aureus (S. aureus) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of complex particles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 8.08 mg/mL, 4.04 mg/mL, and 0 mg/mL). For evaluation of photo-toxicity after 10 minutes, light (intensity 4) was irradiated for 10 minutes by using a Healite II 633 (purchased from Lutronics. Co., Ltd.). The bacteria per concentration after light irradiation were diluted to 1×10.sup.−6 and 1 mL was inoculated in a perifilm (purchased from 3M) and cultured in an incubator (37° C.) for 24 hours, and then the bacterial count was measured.

(61) As a control experiment, an MB aqueous solution corresponding to each concentration of the complex particles was prepared and the bacterial count was measured through the same process as the Example 4 (1) {circle around (5)}.

(62) Further, in order to confirm bacterial toxicity of only the complex particles except for the light source, evaluation of dark-toxicity was performed and illustrated in the Example 4 (2).

(63) (2) Evaluation of Dark-Toxicity Using Staphylococcus Aureus

(64) {circle around (1)} Evaluation of Dark-Toxicity of MBD NPs

(65) In order to evaluate dark-toxicity to bacteria of the complex particles MBD NPs prepared through the Examples 1 and 2 depending on a concentration, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Staphylococcus aureus (S. aureus) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of complex particles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 8.08 mg/mL, 4.04 mg/mL, and 0 mg/mL). The bacteria per concentration were diluted to 1×10.sup.−6 and 1 mL was inoculated in a perifilm (purchased from 3M) and cultured in an incubator (37° C.) for 24 hours, and then the bacterial count was measured. As a control experiment, an MB aqueous solution corresponding to each concentration of the nanoparticles was prepared and the bacterial count was measured through the same process as the Example 4 (2) {circle around (1)}. All of the processes of the Example 4 (2) {circle around (1)} were performed in a dark room where the light was blocked.

(66) {circle around (2)} Evaluation of Dark-Toxicity of MBI NPs

(67) In order to evaluate dark-toxicity to bacteria of the nanoparticles MBI NPs prepared through the Example 1 depending on a concentration, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Staphylococcus aureus (S. aureus) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of nanoparticles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 8.08 mg/mL, 4.04 mg/mL, and 0 mg/mL). The bacteria per concentration were diluted to 1×10.sup.−6 and 1 mL was inoculated in a perifilm (purchased from 3M) and cultured in an incubator (37° C.) for 24 hours, and then the bacterial count was measured. As a control experiment, an MB aqueous solution corresponding to each concentration of the complex particles was prepared and the bacterial count was measured through the same process as the Example 4 (2) {circle around (2)}. All of the processes of the Example 4 (2) {circle around (2)} were performed in a dark room where the light was blocked.

(68) {circle around (3)} Evaluation of Dark-Toxicity of MBT NPs

(69) In order to evaluate dark-toxicity to bacteria of the complex particles MBT NPs prepared through the Examples 1 and 2 depending on a concentration, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Staphylococcus aureus (S. aureus) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of complex particles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 8.08 mg/mL, 4.04 mg/mL, and 0 mg/mL). The bacteria per concentration were diluted to 1×10.sup.−6 and 1 mL was inoculated in a perifilm (purchased from 3M) and cultured in an incubator (37° C.) for 24 hours, and then the bacterial count was measured. As a control experiment, an MB aqueous solution corresponding to each concentration of the complex particles was prepared and the bacterial count was measured through the same process as the Example 4 (2) {circle around (3)}. All of the processes of the Example 4 (2) {circle around (3)} were performed in a dark room where the light was blocked.

(70) {circle around (4)} Evaluation of Dark-Toxicity of MBS NPs

(71) In order to evaluate dark-toxicity to bacteria of the complex particles MBS NPs prepared through the Examples 1 and 2 depending on a concentration, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Staphylococcus aureus (S. aureus) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of complex particles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 4.04 mg/mL, and 0 mg/mL). The bacteria per concentration were diluted to 1×10.sup.−6 and 1 mL was inoculated in a perifilm (purchased from 3M) and cultured in an incubator (37° C.) for 24 hours, and then the bacterial count was measured. As a control experiment, an MB aqueous solution corresponding to each concentration of the complex particles was prepared and the bacterial count was measured through the same process as the Example 4 (2) {circle around (4)}. All of the processes of the Example 4 (2) {circle around (4)} were performed in a dark room where the light was blocked.

(72) {circle around (5)} Evaluation of Dark-Toxicity of MBA NPs

(73) In order to evaluate dark-toxicity to bacteria of the complex particles MBA NPs prepared through the Examples 1 and 2 depending on a concentration, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Staphylococcus aureus (S. aureus) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of complex particles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 8.08 mg/mL, 4.04 mg/mL, and 0 mg/mL). The bacteria per concentration were diluted to 1×10.sup.−6 and 1 mL was inoculated in a perifilm (purchased from 3M) and cultured in an incubator (37° C.) for 24 hours, and then the bacterial count was measured. As a control experiment, an MB aqueous solution corresponding to each concentration of the nanoparticles was prepared and the bacterial count was measured through the same process as the Example 4 (2) {circle around (5)}. All of the processes of the Example 4 (2) {circle around (5)} were performed in a dark room where the light was blocked.

(74) The evaluation of (1) photo-toxicity and (2) dark-toxicity of the Example 4 was illustrated in FIG. 5.

Example 5: Evaluation of Photo-Toxicity for Propionibacterium Acnes (P. Acnes) of Self-Assembled Complex Particles Containing Methylene Blue and Salicylic Acid as Photosensitizer in Aqueous Environment

(75) In order to evaluate photo-toxicity to Propionibacterium acnes of MBS NPs having the highest productivity of singlet oxygen selected from complex particles prepared through the Examples 1 and 2 depending on a concentration in the same light irradiation, 1 mL of a culture medium (LB medium) in which 1×10.sup.6/mL of Propionibacterium acnes (P. Acnes) was dispersed was added in each well of a culture dish (12 well-plate) and then 0.5 mL of complex particles per concentration was added (final concentration of 40.4 mg/mL, 20.2 mg/mL, 4.04 mg/mL, and 0 mg/mL). For evaluation of photo-toxicity after 10 minutes, light (intensity 4) was irradiated for 10 minutes by using a Healite II 633 (purchased from Lutronics. Co., Ltd.). The bacteria per concentration after light irradiation were strained by using Live/Dead® BacLight™ Bacterial Viability Kits and diluted to 1×10.sup.−6/mL, and 200 μl was divided and added in each well of a 96 well-plate and fluorescence-measured (IVIS-Spectrum), and then a fluorescence amount of survival bacteria was calculated by an ROI value of fluorescence imaging. The image and the evaluation of photo-toxicity for Propionibacterium acnes in Example 5 were illustrated in FIGS. 6A and 6B and all of the processes in the Example 5 were performed in a dark room where the light was blocked.

(76) A comparison on effects of a case of using complex particles MBX NPs for phototherapy according to the present invention and a case of using ALA, MAL, and the like as a photosensitive treating agent in the prior arts is illustrated in Table below.

(77) As described in Table below, in the case of treating a skin disease such as acne by using the complex particles for phototherapy according to the present invention, it cannot be seen that a treatment effect is significantly excellent compared with a case of using the ALA, MAL, and the like as a photosensitive treating agent in the prior arts. However, as compared with the treating agent in the prior arts, it is clearly seen that an occlusion time is short and side effects are few, and further, a light protection time for maintaining patient's daily life after treatment is significantly short as compared with ALA, MAL, and the like.

(78) TABLE-US-00001 TABLE 1 Photosensitizer ALA MAL MBX NPs Occlusion time after T > 3 hr T > 3 hr T = ~10 min coating wavelength of light 417 nm 630 nm 633 nm source Treatment effect strong strong mild Side effects Erythema, Erythema, Mild erythema inflammation, inflammation, keratin, keratin, xeroderma xeroderma (damage of (damage of sebaceous glands) sebaceous glands) Light protection time T > 30 hr T > 30 hr X (~0 hr) after operation