PHOTOTHERANOSTIC NANOAGENTS WITH EXCELLENT ATHEROSCLEROTIC PLAQUE-TARGETING AND PLAQUE-PENETRATING PROPERTIES, AND USE THEREOF

Abstract

The present disclosure provides nanoparticles including laminarin and a near-infrared responsive photosensitizer covalently bonded thereto, and a composition for preventing, diagnosing, or treating arteriosclerosis comprising the same as an active ingredient. According to the present disclosure, not only the atherosclerotic plaque targeting and plaque-penetrating properties can be enhanced as compared to conventional photodynamic therapy, thus capable of being usefully used for in vivo imaging of atherosclerotic plaques, but also the size of atherosclerotic plaques is reduced by inducing apoptosis of macrophages in atherosclerotic plaques, thus capable of stabilizing atherosclerotic plaques. Therefore, the composition comprising the nanoparticles of the present disclosure as an active ingredient is expected to be usefully used for the prevention, diagnosis and/or treatment of arteriosclerosis, in that photodynamic therapy and image diagnostic of arteriosclerosis can be performed simultaneously or sequentially.

Claims

1. Nanoparticles comprising a conjugate of laminarin and a near-infrared responsive photosensitizer covalently bonded thereto.

2. The nanoparticles according to claim 1, wherein the laminarin is located outside the nanoparticles and the near-infrared photosensitizer is located inside the nanoparticles.

3. The nanoparticles according to claim 1, wherein the nanoparticles are produced by self-assembly of conjugates.

4. The nanoparticles according to claim 1, wherein the near-infrared responsive photosensitizer is at least one selected from the group consisting of chlorin e6 (Ce6), porphyrin, photofrin, temoporfin, aminolevulinic acid-induced protoporphyrin IX, motexafin lutetium, padoporfin, padeliporfin, talaporfin (NPe.sub.6), radachlorin, Purlytin, phthalocyanines, Verteporfin, HPPH (photochlor), TPC (5-(4-carboxyphenyl)-10,15,20-triphenyl-2,3-dihydroxychlorin), Chlorin p6 (Cp6), Purpurin-18, purpurinimide, and bacteriochlorin.

5. The nanoparticles according to claim 1, wherein the covalent bond is formed between a hydroxyl group of laminarin and a carboxyl group of the photosensitizer.

6. The nanoparticles according to claim 1, wherein the laminarin has an average molecular weight of 1 kDa to 9 kDa.

7. The nanoparticles according to claim 1, wherein the laminarin is contained in an amount of 60 wt % to 95 wt % based on the total weight of the nanoparticle.

8. The nanoparticles according to claim 1, wherein the near infrared responsive photosensitizer is contained in an amount of 5 wt % to 40 wt % based on the total weight of the nanoparticles.

9. The nanoparticles according to claim 1, wherein the nanoparticles have a diameter of 10 to 500 nm.

10. A composition comprising the nanoparticles of claim 1.

11. A method of preventing, diagnosing or treating arteriosclerosis comprising administering to a subject a composition comprising the nanoparticles of claim 1 as an active ingredient.

12. The method according to claim 11, wherein the method of preventing or treating arteriosclerosis further comprises irradiating a near-infrared light after administration of the composition to the subject.

13. The method according to claim 11, wherein the method for diagnosing arteriosclerosis further comprises, irradiating a near-infrared light after administration of the composition to the subject; and measuring the intensity of the near-infrared fluorescence signal emitted from the nanoparticles and comparing it with the result of a normal subject or an arteriosclerosis patient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0116] FIG. 1 is a schematic diagram which shows a method for synthesizing LAM-Ce6 (Laminarin-chlorin e6) using laminarin and chlorin e6 and a crosslinking agent EDC/DMAP, and a state in which LAM-Ce6 forms self-assembled nanoparticles on an aqueous solution.

[0117] FIG. 2 is a particle size distribution of LAM-Ce6 according to an embodiment of the present disclosure (a), is a diagram showing a scanning electron microscope image of LAM-Ce6 (scale bar: 500 nm) (b).

[0118] FIG. 3 shows the Fourier transform infrared spectral spectrum of laminarin (red) (a), shows the Fourier transform infrared spectral spectrum of LAM-Ce6 (blue) (b), and shows the Fourier transform infrared spectral spectrum of Ce6 (black) (c).

[0119] FIGS. 4a, 4b and 4c show the optical characteristics. FIG. 4a shows the ultraviolet and visible spectrum of Ce6 and LAM-Ce6 dissolved in PBS containing 1% Tween 20, and LAM-Ce6 dispersed in PBS. FIG. 4b shows the fluorescence spectra of Ce6 and LAM-Ce6 dissolved in PBS containing 1% Tween 20, and LAM-Ce6 dispersed in PBS. FIG. 4c shows the results of singlet oxygen generation over time after irradiating 670 nm to Ce6 and LAM-Ce6 dissolved in PBS containing 1% Tween 20, and LAM-Ce6 dispersed in PBS.

[0120] FIG. 5 shows the results of examining the cytotoxicity through CCK-8 kit at various concentrations of LAM-Ce6 based on Ce6 for macrophages, activated macrophages and foam cells (a) and shows the results of examining the phototoxicity through CCK-8 kit when each cell is treated with various concentrations of Ce6 standards and then irradiated with a 670 nm laser (b).

[0121] FIG. 6a shows a fluorescence image comparing the intracellular absorption of LAM-Ce6 to macrophages, activated macrophages, and foam cells.

[0122] FIG. 6b shows fluorescence images comparing the degree of intracellular absorption of LAM-Ce6 after first being treated with laminarin serving as an agonist and β-glucan serving as an antagonist in order to compare the ligand-receptor mediated endocytosis by Dectin-1.

[0123] FIG. 7 shows in vivo fluorescence images acquired through a small animal imaging device for each time in order to evaluate the distribution and excretion of LAM-Ce6 over time after intravenous injection of LAM-Ce6 into Balb/c nude mice (a) and shows the ex vivo tissue distribution of LAM-Ce6 through fluorescence analysis in liver, lung, spleen, kidney and heart extracted from mice 48 hours after LAM-Ce6 administration (b).

[0124] FIG. 8 shows the results of examining the concentrations of alanine aminotransferase (ALP), aspartate aminotransferase (AST), alkaline phosphatase (ALT) and blood uric acid (SUA) in blood by collecting blood 24 hours after intravenous injection of LAM-Ce6.

[0125] FIG. 9 shows the results of phototoxicity in skin tissue and H&E staining of dissected skin tissue after 3 days, in a state where the skin was irradiated with a 670 nm laser one hour after intravenous injection of LAM-Ce6 and Ce6 (equiv. of Ce6 5 μM),

[0126] FIG. 10 is a schematic diagram of the molecular imaging experimental method protocol for in vivo mouse atherosclerotic plaque target imaging of LAM-Ce6(a) and is a schematic diagram of a multichannel fluorescence microscope in vivo molecular imaging system (Customized multichannel IVFM imaging system) (b).

[0127] FIG. 11 shows the results of fluorescence images acquired by an in vivo molecular imaging system of the carotid artery region under a multi-channel fluorescence microscope 48 hours after intravenous injection of LAM-Ce6 4 mg/kg (equiv. of Ce6) (a) and is a diagram showing the result of a fluorescent image of a tissue cross-section in which the extracted carotid artery was sectioned to 10 μm (b).

[0128] FIG. 12 is a schematic diagram of a protocol for viewing the therapeutic effect of LAM-Ce6 nanoparticles by photodynamic therapy of mouseatherosclerotic plaques.

[0129] FIG. 13 is the fluorescence image results of atherosclerotic plaques acquired with a multi-channel fluorescence microscope in vivo molecular imaging system one week after irradiating a laser (670 nm, 1 W/cm.sup.2, 100 J/cm.sup.2) to the carotid artery atherosclerotic plaque 48 hours after intravenous injection of LAM-Ce6 4 mg/kg (equiv. of Ce6) (a), is a diagram showing a fluorescence image of a tissue cross-section in which the extracted carotid artery was sectioned to 10 μm and an image stained with F4/80, Dectin-1, H&E, and ORO (b).

[0130] FIG. 14 is a schematic diagram of an FRI imaging method protocol for in vivo rabbit atherosclerotic plaque target imaging of LAM-Ce6 nanoparticles.

[0131] FIG. 15 is a schematic diagram of an OCT-NIRF system and an intravascular imaging system for in vivo imaging using LAM-Ce6 nanoparticles in a rabbit arteriosclerosis model.

[0132] FIG. 16 shows the image results acquired from the aortic atherosclerotic plaque 48 hours after the injection of 4 mg/kg (equiv. of Ce6) of LAM-Ce6 nanoparticles into the ear vein in a rabbit arteriosclerosis model (a) and is a diagram showing the results of a fluorescence image of a tissue cross-section in which the extracted carotid artery was sectioned to 10 μm (b).

[0133] FIG. 17 is a schematic diagram of a protocol for observing the therapeutic effect of LAM-Ce6 nanoparticles by photodynamic therapy of rabbit arteriosclerotic plaque.

[0134] FIG. 18 is fluorescence images obtained from aortic atherosclerotic plaques 48 hours after injection of LAM-Ce6 nanoparticles 4 mg/kg (equiv. of Ce6) into the ear vein (uptake) and after photodynamic therapy (after PDT) in a rabbit arteriosclerosis model (a), is a fluorescence image obtained in the forward and flip directions in the arteriosclerotic plaque after photodynamic therapy (b) and is a view showing a fluorescence image and a TUNEL staining image of a tissue cross-section in which the carotid artery extracted according to laser irradiation was sectioned to 10 μm (c).

[0135] FIG. 19 shows the results of histological analysis of the degree of atherosclerotic plaque retraction and stabilization through phototherapy. (a) shows that the inflammatory signal was greatly reduced after 4 weeks of phototherapy progress. (b) shows the results of measurement of RAM11 expression level showing the degree of reduction in the size of macrophages and arteriosclerotic plaques, and PSR staining images showing the amount of collagen.

DETAILED DESCRIPTION

[0136] Hereinafter, the present disclosure will be described in more detail with reference to examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present disclosure as set forth in the appended claims is not limited to or by the examples.

Preparation Example: Synthesis of Dectin-1 Target Nanoparticles (LAM-Ce6 Nanoparticles)

[0137] LAM-Ce6 nanoparticles for targeting Dectin-1 expressed in atherosclerotic plaques were synthesized as shown in the following Reaction Scheme.

##STR00003##

[0138] Laminarin (Sigma-Aldrich) (0.1 g) was completely dissolved in 20 mL of DMSO at 60° C. for 24 hours. EDC (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, Sigma-Aldrich) (0.0213 g), DMAP (4-Dimethylaminopyridine, Sigma-Aldrich) (0.0136 g) and Ce6 (Chlorin e6, Frontier Scientific) (0.0333 g) were dissolved in the dissolved laminarin solution, and the reaction was carried out. EDC and DMAP were further added twice, and the reaction was carried out for 2 days. The solution obtained after the reaction was dialyzed in 50% DMSO aqueous solution (dimethyl sulfoxide, Samjeon Chemical and Sigma-Aldrich) for 5 days using a regenerated cellulose dialysis membrane (1 kD Molecular weight cut-off (MWCO), Spectrum Laboratories) for 5 days, and further dialyzed for 2 days, purified and then lyophilized (see FIG. 1). The result was referred to as LAM-Ce6 (Laminarin-Ce6). LAM represents the laminarin moiety and Ce6 represents the chlorine e6 moiety.

Example 1: Characterization of LAM-Ce6 nanoparticles

[0139] 1.1: Confirmation of Size, Polydispersity Index, Z-Average Size and Morphology of LAM-Ce6 Nanoparticles

[0140] 1.1.1: Nanoparticle Size, Polydispersity Index and Z-Average Size

[0141] The dried LAM-Ce6 was dispersed in distilled water at 0.1 mg/mL, and then analyzed with a particle size analyzer (dynamic light scattering, DLS).

[0142] 1.1.2: Nanoparticle Morphology

[0143] 1 mg of LAM-Ce6 was dispersed in distilled water, and the dispersed solution was dropped on a glass cover slip, allowed the sample to air dry and then coated with platinum. The analysis was performed using a field emission scanning electron microscope (FE-SEM) operating at 15.0 kV. The results are shown in FIG. 2(a) and FIG. 2(b).

[0144] 1.1.3: Results

[0145] As shown in FIG. 2, the Z-average size of LAM-Ce6 dispersed in distilled water is 137 nm, the average size of the particles is 144.9±34.5 nm, and the polydispersity index (PDI) is 0.135 (see FIG. 2(b)). In addition, it was confirmed that LAM-Ce6 has a spherical shape (see FIG. 2(b)). This is a result proving that when LAM-Ce6 exists in an aqueous solution, hydrophilic LAM is rearranged outside the particle and hydrophobic Ce6 is re-arranged inside the particle to form a self-assembled nanostructure.

[0146] 1.2: Confirmation of Binding of LAM and Ce6 of LAM-Ce6 Nanoparticles

[0147] Laminarin, resulting LAM-Ce6 and Ce6, was analyzed by Fourier transform infrared spectroscopy (FT-IR). For FT-IR analysis, samples were prepared in the form of potassium bromide (KBr) pellets. FT-IR spectra were acquired with a resolution of 4 cm.sup.−1 in the range of 4,000 cm.sup.−1 to 400 cm.sup.−1.

[0148] The results are shown as laminarin (red) in FIG. 3(a), LAM-Ce6 (blue) in FIG. 3 (b), and Ce6 (black) in FIG. 3 (c), respectively.

[0149] The peak of the N—H bond appearing at 2961 cm.sup.−1 and the peak of the C═H bond appearing at 1709 cm.sup.−1 of the Ce6 spectrum shown as Ce6 (black) in FIG. 3(c) appear at 2947 cm.sup.−1 and 1684 cm.sup.−1 of the LAM-Ce6 spectrum shown as LAM-Ce6 (blue) in FIG. 3(b). The result proves that LAM and Ce6 are properly bound.

[0150] 1.3: Confirmation of Structure Formation of LAM-Ce6 Nanoparticles

[0151] UV/Vis (ultraviolet and visible light) and fluorescence spectra were analyzed to determine whether LAM-Ce6 forms nanoparticles. For analysis, LAM-Ce6 was dissolved in PBS so as to be 5 μM based on Ce6, and for comparison, LAM-Ce6 and Ce6 were prepared in PBS containing 1% Tween 20 at the same concentration. UVNis absorbance was acquired from 300 nm to 800 nm, and the fluorescence intensity was acquired from 600 nm to 800 nm.

[0152] The results are shown in FIG. 4a and FIG. 4b.

[0153] As shown in FIG. 4a and FIG. 4b, the absorbance and fluorescence intensity of Ce6 (PBS with 1% Tween 20) and LAM-Ce6 (PBS with 1% Tween 20) were similar to each other, whereas both the absorbance and fluorescence intensity of LAM-Ce6 (PBS) showed low results. The LAM-Ce6 (PBS) of the present disclosure exhibited reduced UVNis wavelength absorbance and fluorescence intensity, as compared to Ce6 (PBS with 1% Tween 20) and LAM-Ce6 (PBS with 1% Tween 20), in which the formation of self-assembled nanoparticles was inhibited by a surfactant, whereby it can be confirmed that the LAM-Ce6 (PBS) of the present disclosure forms the nanoparticle that the Ce6 is located inward. That is, from the above results, it was verified that Ce6 forms a LAM-Ce6 nanostructure that the Ce6 is located inward in an aqueous solution.

[0154] 1.4: Confirmation of Singlet Oxygen Generation of LAM-Ce6 Nanoparticles

[0155] The singlet oxygen sensor green (SOSG, Invitrogen) fluorescence intensity was analyzed to determine whether singlet oxygen was generated when irradiating LAM-Ce6 with a laser having a near-infrared light (670 nm wavelength). For analysis, PBS at pH 7.4 was saturated with oxygen for 30 min and LAM-Ce6 was dissolved at 5 μM based on Ce6. For comparison, Ce6 and LAM-Ce6 were dissolved in oxygen-saturated PBS containing 1% Tween 20 at the same concentration. All three solutions contained 1 μM SOSG. The fluorescence intensity of SOSG (Ex(excitation)/Em(emission): 504 nm/525 nm) was measured by irradiating a laser (670 nm, 50 mW/cm.sup.2) every 30 seconds from 0 to 120 seconds.

[0156] The results are shown in FIG. 4c.

[0157] As shown in FIG. 4c, the SOSG fluorescence intensity of Ce6 (PBS with 1% Tween 20) and LAM-Ce6 (PBS with 1% Tween 20) showed the tendency to increase similarly to each other, whereas LAM-Ce6 (PBS) showed a relatively low fluorescence intensity. That is, the LAM-Ce6 (PBS) of the present disclosure generates singlet oxygen when irradiated with a near-infrared light, but it generates significantly lower singlet oxygen as compared to Ce6 (PBS with 1% Tween 20) and LAM-Ce6 (PBS with 1% Tween 20), in which the formation of self-assembled nanoparticles was inhibited by the surfactant. Therefore, the above results suggest that the exposure of singlet oxygen to the non-target is low when irradiated with a near-infrared light after injection into the body.

[0158] The present inventors were able to prove from the above results that LAM-Ce6 forms nanoparticles, and demonstrated that when irradiated with a near-infrared light to LAM-Ce6, singlet oxygen was generated and the effect of phototoxicity was small during circulation in the body.

Example 2. Evaluation of Cytotoxicity and Phototoxicity of LAM-Ce6 Nanoparticles

[0159] 2.1: Evaluation of Cytotoxicity of LAM-Ce6 Nanoparticles

[0160] The in vitro cytotoxicity of LAM-Ce6 against macrophages, activated macrophages and foam cells was evaluated using a CCK-8 assay kit (Cell Counting Kit-8, Dojindo Molecular Technologies, Inc.).

[0161] Specifically, RAW 264.7 cells (monocytes and macrophages of mouse BALB/c, Korea Cell Line Bank, KCLB, No. 40071) were cultured in RPMI 1640 (Roswell Park Memorial Institute 1640 media, Welgene Inc.) containing 10% FBS and 1% penicillin-streptomycin at 37° C. Subcultured RAW 264.7 cells (2×10.sup.4 cells/well) were transferred to 96-well culture plates and allowed to adhere for 1 day. To prepare activated macrophages and foam cells, lipopolysaccharide (LPS, Sigma-Aldrich) (200 ng/mL) and low-density lipoprotein (LDL, Sigma-Aldrich) (100 μg/mL) were treated for 1 day. Macrophages, activated macrophages and foam cells were treated with various concentrations of LAM-Ce6 (1 μM, 2 μM, 5 μM, 10 μM and 20 μM) for 4 hours. After 2 hours of material treatment, CCK-8 solution was added to the cells, and the cells were further cultured for 2 hours, and the absorbance was measured at 450 nm.

[0162] 2.2: Evaluation of Phototoxicity of LAM-Ce6 Nanoparticles

[0163] A near-infrared laser (670 nm, 50 mW/cm.sup.2) was irradiated after cell culture and material treatment in the same manner as in Example 2.1. After 16 hours, CCK-8 solution was added to the cells, and the cells were further cultured for 2 hours, and then the absorbance was measured at 450 nm.

[0164] 2.3: Results

[0165] FIG. 5(a) shows the cytotoxicity evaluation results, and FIG. 5(b) shows the phototoxicity evaluation results.

[0166] When the near-infrared laser was not irradiated as shown in FIG. 5(a), the cell viability (%) according to the LAM-Ce6 concentration was not significantly different from the case where LAM-Ce6 was not added in all cells. This means that LAM-Ce6 itself has no toxicity against macrophages, activated macrophages and foam cells.

[0167] However, when irradiated with a near-infrared laser as shown in FIG. 5 (b), the viability of macrophages, activated macrophages and foam cells was decreased in a concentration-dependent manner. From the above results, the inventors confirmed that LAM-Ce6 is phototoxic to these cells.

Example 3. Confirmation of Intracellular Absorption of LAM-Ce6 Nanoparticles

[0168] It is necessary to confirm whether laminarin modified by binding to Ce6 also absorbs LAM-Ce6 nanoparticles into cells through Dectin-1 and ligand-receptor binding.

[0169] Concentration-Dependent Intracellular Absorption of LAM-Ce6

[0170] To evaluate the intracellular absorption of LAM-Ce6, RAW 264.7 cells (1×10.sup.5 cells/well) were cultured in 4-well chamber slides. Next, activated macrophages and foam cells were established by treatment with LPS and LDL. When macrophages were treated with LPS and LDL, macrophages were activated, and the activated macrophages were differentiated to become foam cells. Macrophages, activated macrophages and foam cells were treated with LAM-Ce6 (1 μM, 5 μM and 10 μM) and incubated for 4 hours. Then, the cells were washed 3 times with DPBS and was fixed with 3.7% formalin for 30 minutes. The fixed cells were treated with DAPI to stain the cell nuclei. Then, the cells were imaged using a fluorescence microscope.

[0171] The results are shown in FIG. 6a.

[0172] As shown in FIG. 6a, the concentration-dependent NIRF (near-infrared fluorescence) signal of LAM-Ce6 was increased in activated macrophages and foam cells as compared to macrophages. That is, NIRF (near-infrared fluorescence) signal increased in a concentration-dependent manner of LAM-Ce6 in activated macrophages and foam cells as compared to macrophages. That is, the LAM-Ce6 nanoparticles according to the invention were selectively absorbed by activated macrophages and foam cells as compared to non-activated macrophages.

[0173] Ligand-Receptor Mediated Intracellular Absorption of LAM-Ce6

[0174] To evaluate the ligand-receptor mediated intracellular absorption of LAM-Ce6, RAW 264.7 cells (1×10.sup.5 cells/well) were cultured in 4-well chamber slides. Next, activated macrophages and foam cells were established by treatment with LPS and LDL. Laminarin (1 mg/mL) acting as an agonist and β-glucan acting as an antagonist (1 mg/m L, β-1,3-Glucan from Euglena gracilis, Sigma Aldrich, CAS Number: 9051-97-2) against macrophages, activated macrophages and foam cells were treated for 1 hour each, then treated with LAM-Ce6 (1 μM, 5 μM, and 10 μM) and incubated for 4 hours. Cells were washed with DPBS, fixed with 3.7% formalin, and then the cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, Dana Korea and GBI Labs).

[0175] The results are shown in FIG. 6b.

[0176] As shown in FIG. 6b, the fluorescence signal of LAM-Ce6 increased when pre-treated with laminarin, whereas the fluorescence signal of LAM-Ce6 decreased when pre-treated with β-glucan.

[0177] These results were resulted from the activation of ligand-receptor mediated endocytosis by Dectin-1, resulting in increased intracellular absorption of LAM-Ce6 when pre-treated with laminarin, and resulted from the inhibition of ligand-receptor-mediated endocytosis by Dectin-1, resulting in decreased intracellular absorption of LAM-Ce6 when pre-treated with 1-glucan.

[0178] From the above results, the present inventors demonstrated that LAM-Ce6 was absorbed into cells by Dectin-1.

Example 4. Evaluation of Biodistribution and Excretion of LAM-Ce6 Nanoparticles

[0179] To evaluate the distribution and excretion of LAM-Ce6 in the body over time, Balb/c mice were injected with LAM-Ce6 (2 mg/kg/100 μL) at 2 mg/kg of mouse body weight via tail vein. In vivo near-infrared fluorescence images were acquired using an imaging device for small animals at 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours and 48 hours after injection.

[0180] The results are shown in FIG. 7(a).

[0181] As shown in FIG. 7(a), a strong NIRF signal was observed up to 6 hours after LAM-Ce6 injection, after which the signal intensity gradually decreased. However, the NIRF signal remained visible for up to 2 days, which is a result demonstrating that the circulation period in the body of nanoparticles was extended.

[0182] In addition, 48 hours after LAM-Ce6 administration, liver, lung, spleen, kidney and heart, known as organs in which a large number of macrophages exist, were extracted from Balb/c mice, and ex vivo fluorescence images were acquired and analyzed.

[0183] The results are shown in FIG. 7(b).

[0184] As shown in FIG. 7(b), 48 hours after LAM-Ce6 injection, the NIRF signal was still detected in liver tissue due to the amphiphilic nature of LAM-Ce6 nanoparticles.

[0185] From the above results, it can be confirmed that when upon injection of the LAM-Ce6 nanoparticles of the present disclosure, nanoparticles circulate throughout the body for up to 6 hours and start to discharge after 6 hours, exist in the body of the mouse for at least 2 days, are distributed in the lungs, spleen and kidney tissues in the body, and particularly, are mainly distributed in liver tissue.

Example 5. Confirmation of the Presence of Liver Function Damage by LAM-Ce6 Nanoparticles

[0186] From Example 4, it was confirmed that LAM-Ce6 nanoparticles were mainly distributed in liver tissue when injected into the body. Thus, whether LAM-Ce6 nanoparticles cause damage to liver function was confirmed by blood concentration analysis of Alanine aminotransferase (ALT), aspartate amiotransaminase (AST), Alkaline aminophosphatase (ALP) and Serum uric acid (SUA) 24 hours after LAM-Ce6 injection.

[0187] To analyze the concentrations of ALP, AST, ALT and SUA in blood after LAM-Ce6 injection, 2 mg LAM-Ce6 per body weight(kg) (2 mg/kg/100 μL), 4 mg LAM-Ce6 per body weight(kg) (4 mg/kg/100 μL) and 6 mg of LAM-Ce6 per body weight(kg) (6 mg/kg/100 μL) (equiv. of Ce6) were injected into 8-week-old Balb/c mice by tail vein injection. 24 hours after the drug injection, blood was collected through the vena cava. The serum was obtained by centrifugation (1500×g, 10 min, 4° C.). The concentrations of ALP, AST, ALT and SUA in serum were measured through blood chemistry assay.

[0188] The results are shown in FIG. 8.

[0189] As shown in FIG. 8, at all LAM-Ce6 injection concentrations, ALP, AST, ALT, and SUA were all within the normal range (yellow box) of concentrations. This is the result of confirming that the LAM-Ce6 nanoparticles of the present disclosure, which are mainly distributed in liver tissue during intravenous injection, do not cause significant damage to liver function.

Example 6. Evaluation of Skin Phototoxicity of LAM-Ce6 Nanoparticles

[0190] To evaluate the skin phototoxicity of LAM-Ce6, 8-week-old Balb/c mice were intravenously injected with 2 mg of LAM-Ce6 (2 mg/kg/100 μL) per body weight (kg) (equiv. of Ce6) and the same concentration of free Ce6 (prepared by dissolving Ce6 in a small amount of DMSO and adding PBS) as a control group. One hour after injection, a near-infrared (670 nm) laser (50 mW/cm.sup.2) was irradiated for 5 minutes. Where erythema occurred on the 2.sup.nd and 3.sup.rd days was confirmed and the skin tissue was peeled off and H&E (hematoxylin and eosin) staining was performed.

[0191] The results are shown in FIG. 9.

[0192] As shown in FIG. 9, since the erythema occurred in the mouse injected with free Ce6 (free Ce6), unlike injected with LAM-Ce6 (LAM-Ce6), it can be confirmed that the LAM-Ce6 did not cause skin phototoxicity because it exited in the form of nanoparticles, unlike free Ce6.

Example 7. LAM-Ce6 Nanoparticles Target Macrophages in Atherosclerotic Plaques in Mice In Vivo

[0193] To evaluate the properties of LAM-Ce6 against macrophage targets in atherosclerotic plaques, an in vivo molecular imaging system was used in a mouse model in which arteriosclerosis was induced. FIG. 10 (a) shows the experimental method protocol, and FIG. 10 (b) shows a schematic diagram of a customized multi-channel in vivo molecular imaging system.

[0194] Atherosclerosis Mouse Model Formation and LAM-Ce6 Nanoparticle Injection Method

[0195] Similar to the protocol shown in FIG. 10(a), a high-cholesterol diet (HCD) was supplied to 7-week-old ApoE.sup../. genetically modified mice (C.KOR/StmSlc-Apoe.sup.shl, imported from Japan SLC, purchased from Central Laboratory Animals Co., Ltd.) for a total of 10 weeks to produce atherosclerotic plaques. 48 hours after injection of LAM-Ce6 (4 mg/kg) (equiv. of Ce6) into the tail vein, signals in the bilateral carotid arteriosclerotic plaques of mice were measured through multichannel fluorescence microscopy in vivo molecular imaging (IVFM) in the atherosclerotic plaques. After obtaining the IVFM image, the carotid artery was extracted and sectioned into a size of 10 μm, and then a fluorescence image of the tissue section (ex vivo FM imaging) was obtained.

[0196] Multi-Channel Fluorescence Microscopy In Vivo and Ex Vivo Fluorescence Imaging Methods

[0197] In addition, as shown in the schematic diagram of FIG. 10(b), molecular imaging was performed through a customized multichannel IVFM imaging system. Specifically, in the imaging system, several laser wavelengths are generated by an excitation module and irradiated to atherosclerotic plaques through an optical system to activate LAM-Ce6. The laser emitted from LAM-Ce6 in the atherosclerotic plaque was delivered to the emission module through the same optical system, and the individual laser lights were separated and measured.

[0198] The IVFM image is shown in FIG. 11(a), and the fluorescence image of the tissue section is shown in FIG. 11(b).

[0199] Confirmation of Absorption Rate of LAM-Ce6 Nanoparticles in Arteriosclerotic Plaque

[0200] As shown in FIG. 11(a), 48 hours after LAM-Ce6 injection, the Ce6 near-infrared signal (red) was measured to be very high in the atherosclerotic plaque (region within the dotted line). Accordingly, it was confirmed that the absorption rate of LAM-Ce6 in the atherosclerotic plaque was high. The FITC (Fluorescein isothiocyanate) shows an angiogram of the carotid artery where autofluorescence occurred at the wavelength corresponding to FITC, the Plaque Mac represents macrophages of sclerotic plaques, and the Merge shows the overlay of fluorescence images of FITC and Laminarin-Ce6.

[0201] As shown in FIG. 11(b), in the image obtained from the cross-section of the atherosclerotic plaque in which the extracted carotid artery was sectioned to 10 μm, it was confirmed that the near-infrared signal (red) of LAM-Ce6 penetrated up to a deep place into the plaque. The autofluorescence represents the intrinsic fluorescence signal of carotid elastic fibers emitted by the FITC signal, and the LAM-Ce6 shows a fluorescence signal by near-infrared rays. The Merge represents the overlay of two fluorescence signals.

Example 8. In Vivo Mouse Atherosclerotic Plaque Photodynamic Therapeutic Effect of LAM-Ce6 Nanoparticles

[0202] A protocol for confirming the effect of photodynamic therapy (PDT) on atherosclerotic plaques is shown in FIG. 12.

[0203] In Vivo Image Analysis of Photodynamic Therapeutic Effects

[0204] To evaluate the photodynamic therapeutic effect of LAM-Ce6 in atherosclerotic plaques, atherosclerotic plaques were generated in 7-week-old ApoE.sup../. transgenic mice through a high-cholesterol diet (HCD) for a total of 10 weeks. Then, the in vivo imaging was performed 48 hours after LAM-Ce6 (4 mg/kg) (equiv. of Ce6) was injected into the tail vein. A laser (670 nm, 1 W/cm 2, 100 J/cm.sup.2) was irradiated to the atherosclerotic plaque. On day 5, in vivo imaging was performed 48 hours after LAM-Ce6 (4 mg/kg) (equiv. of Ce6) was injected into the tail vein (1 week after laser irradiation).

[0205] The results are shown in FIG. 13 (a).

[0206] As shown in FIG. 13 (a), the size of atherosclerotic plaques decreased one week (1 week) after laser irradiation as compared to before (baseline) laser irradiation (see Bright field), and the NI RF signal also decreased (see LAM-Ce6).

[0207] From the above results, the present inventors found that the photoactivation of LAM-Ce6 can restore atherosclerotic plaque, and the inflammation of atherosclerotic plaques can be reduced one week after laser irradiation.

[0208] Histological Analysis of Phototherapy Effect

[0209] To histologically analyze the effect of phototherapy, the extracted carotid artery was sectioned to 10 μm to confirm the fluorescence image, and F4/80, Dectin-1, H&E and ORO staining was performed. The F4/80 is a membrane protein known as a marker of mature mouse macrophages, and antibodies to F4/80 were obtained from Santa Cruz Biotechnology (SC-52664, Santa Cruz Biotechnology). The ORO (Oil Red 0) is a lysochrome diazo dye used for staining triglycerides and lipids, and was obtained from ScyTek (Oil Red 0 Stain Kit (For Fat), ORK-2, LOT #: 57902, ScyTek).

[0210] The results are shown in FIG. 13(b).

[0211] As shown in FIG. 13(b), the fluorescence signal decreased in the subjects treated with LAM-Ce6 light as compared to the control group, which was a group injected only with LAM-Ce6 nanoparticles without irradiating a laser (see FM), and Dectin-1 and ORO stained areas were also reduced as compared with the control group.

[0212] Overall, from the results, the inventors confirmed that LAM-Ce6 photoactivation can effectively attenuate macrophage-mediated inflammatory response and convert plaques to a good stable state after 1 week of phototherapy.

Example 9. LAM-Ce6 Nanoparticles Target Macrophages in Rabbit Atherosclerotic Plaque In Vivo

[0213] To evaluate the properties of LAM-Ce6 nanoparticles against macrophage targets in atherosclerotic plaques, the OCT-NIRF was used in a rabbit model in which atherosclerosis was induced. FIG. 14 shows the experimental method protocol, and FIG. 15 shows a schematic diagram of the OCT-NIRF system.

[0214] Atherosclerosis Rabbit Model Formation and LAM-Ce6 Nanoparticle Injection Method

[0215] Similar to the protocol shown in FIG. 14, a 1% cholesterol diet (HCD) was fed to a NZW rabbit for 1 week, and then the aorta was subjected to balloon injury. After maintaining a 1% cholesterol diet for 3 weeks, 0.1% cholesterol diet was fed for 11 weeks to induce atherosclerotic plaque. The aorta was extracted 48 hours after injecting 4 mg/kg of LAM-Ce6 nanoparticles (equiv. of Ce6) into the ear vein. Signals in rabbit aortic plaques were measured by FRI. Further, after sectioning the arteriosclerotic plaque with 10 μm from the extracted carotid artery, fluorescence images of tissue sections (ex vivo FM imaging) were obtained.

[0216] The results are shown in FIG. 16.

[0217] Confirmation of Absorption Rate of LAM-Ce6 Nanoparticles in Arteriosclerotic Plaque

[0218] As shown in FIG. 16(a) and FIG. 16(b), the Ce6 near-infrared signal (red) was measured to be very high in atherosclerotic plaques 48 hours after LAM-Ce6 nanoparticle injection. Thereby, it was confirmed that the absorption rate of LAM-Ce6 nanoparticles in the atherosclerotic plaque was high.

[0219] As shown in FIG. 16(b), it can be confirmed that in the image obtained from the cross-section of the atherosclerotic plaque in which the extracted carotid artery was sectioned at 10 μm, the near-infrared signal (red) of LAM-Ce6 nanoparticles penetrated up to a deep place into the arteriosclerotic plaque. The autofluorescence refers to the intrinsic fluorescence signal of carotid elastic fibers emitted by the FITC signal, the LAM-Ce6 shows a fluorescence signal by near-infrared rays, and the Merge represents the overlay of two fluorescence signals.

Example 10. In Vivo Rabbit Atherosclerotic Plaque Photodynamic Therapy Effect of LAM-Ce6 Nanoparticles

[0220] To confirm the photodynamic therapy (PDT) effect of LAM-Ce6 nanoparticles of the present disclosure on atherosclerotic plaques in a rabbit model, experiments were performed as in the protocol shown in FIG. 17.

[0221] In Vivo Image Analysis of Photodynamic Therapy Effects

[0222] Similar to the protocol shown in FIG. 17, a 1% cholesterol diet (HCD) was fed to a NZW rabbit for 1 week, and then the aortas were subjected to balloon injury. Then, a 1% cholesterol diet was maintained for 3 weeks, and then a 0.1% cholesterol diet was fed for 11 weeks, thereby generating atherosclerotic plaques. Atherosclerotic plaques in rabbits were imaged through OCT-NIRF at atherosclerotic plaques 48 hours after injection of LAM-Ce6 nanoparticles (4 mg/kg) (equivalent to Ce6) into the ear vein. To proceed with photodynamic therapy, a laser diffuser (670 nm, 500 mW/cm, 150 J/cm) was put into the aorta and located on the atherosclerotic plaque, and then the laser was irradiated. After irradiation, imaging was performed through OCT-NIRF.

[0223] The imaging results are shown in FIG. 18(a) and FIG. 18(b).

[0224] As shown in FIG. 18(a) and FIG. 18(b), only in the laser-irradiated area, the NIRF signal decreased after laser irradiation as compared to before (baseline) laser irradiation. From the above results, the effect of photodynamic therapy that reduces the size of the arteriosclerotic plaque was confirmed.

[0225] OCT-NIRF In Vivo Fluorescence Imaging Method

[0226] Further, similar to the schematic diagram shown in FIG. 15, molecular imaging was performed through the OCT-NIRF system. Briefly, in the imaging system, when the light from the OCT laser light source was transmitted to the lens of the catheter through the core of the optical fiber, the signal reflected from the blood vessel was converted into an electrical signal, so that the shape of the blood vessel could be confirmed. In the NIRF system, when laser with 660 nm wavelength was transferred to a catheter through the core of the optical fiber, the LAM-Ce6 nanoparticles were photoactivated, and the emitted fluorescence was received back into the catheter, and then converted into an electrical signal, thereby confirming the intensity of the fluorescence.

[0227] Histological Analysis of Phototherapy Effect

[0228] To histologically analyze the effect of phototherapy, one hour after laser irradiation, the extracted carotid artery was sectioned to 10 μm, and the fluorescence image was confirmed. To confirm apoptosis by singlet oxygen, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining was performed. TUNEL is a staining method for confirming apoptosis that was used to detect DNA breakage formed when DNA fragmentation occurred in the final stage of apoptosis, and was obtained from Merck.

[0229] The results are shown in FIG. 18(c).

[0230] As shown in FIG. 18(c), LAM-Ce6 nanoparticles treatment and light treatment (LAM-Ce6+Laser) reduced the fluorescence signal in some areas as compared to controls treated with LAM-Ce6 nanoparticles alone, and TUNEL staining was also increased as compared to a control group.

[0231] Overall, from the above results, the inventors confirmed that photoactivation of LAM-Ce6 nanoparticles in atherosclerotic plaques in rabbits can induce apoptosis.

[0232] In Vivo Imaging and Histological Analysis of Atherosclerotic Plaque Retraction and Stabilization Through Phototherapy

[0233] The RAM11 PSR and ORO stainings were performed for histological analysis of atherosclerotic plaque retraction and stabilization through phototherapy. The RAM11 is an antibody capable of labeling rabbit macrophages, and was obtained from Dako. The PSR (Picro-Sirius Red) is a dye that can stain collagen, and was obtained from ScyTek (Picro-Sirius Red Stain Kit (For Collagen), PSR-1, ScyTek).

[0234] The results are shown in FIG. 19.

[0235] As shown in FIG. 19 (a), it was confirmed that the inflammatory signal was greatly reduced after 4 weeks of phototherapy in the area where the inflammatory signal was strong. As shown in FIG. 19(b), when compared with the control group not irradiated with the laser, it was confirmed that the macrophage and the size of atherosclerotic plaques was reduced through the decrease in the expression of RAM11. It was confirmed through PSR staining that the amount of collagen was increased compared to the control group.

[0236] Overall, from the above results, the inventors confirmed that photoactivation of LAM-Ce6 nanoparticles reduces the number of macrophages in rabbit arteriosclerotic plaques, allowing the atherosclerotic plaques to regress, and the increase in collagen can convert the atherosclerotic plaques into a good stable state.

CONCLUSION

[0237] The present disclosure provides novel photoactivated theranostic nanoparticles that specifically bind to macrophage Dectin-1, for example, LAM-Ce6 nanoparticles according to an embodiment of the present disclosure. LAM-Ce6 nanoparticles exhibited negligible toxicity in the absence of light, and caused a large amount of macrophage apoptosis upon laser irradiation on macrophages. In addition, LAM-Ce6 nanoparticles were evaluated to have no toxicity in blood chemistry analysis and skin phototoxicity analysis. Moreover, the photoactivated theranostic nanoparticles of the present disclosure enable in vivo imaging of atherosclerotic plaque macrophages.

[0238] Therefore, when using the composition comprising, as an active ingredient, the nanoparticles comprising the near-infrared reactive photosensitizer covalently bonded to laminarin according to the present disclosure, it specifically binds to Dectin-1 of macrophages in atherosclerotic plaques and can be imaged by generating a near-infrared fluorescence signal only when irradiated with a near-infrared laser, and thus can be useful for imaging diagnostics. Further, it can induce apoptosis of atherosclerotic macrophages to stabilize the atherosclerotic plaque, reduce its size and reduce the macrophage-mediated inflammatory response, which can be usefully used for the prevention or treatment of arteriosclerosis.

[0239] The photoactivatable LAM-Ce6 nanoparticle fusion material of the present disclosure can targeting Dectin-1 present disclosure is expected to be promisingly used for selective fluorescence imaging and PDT in terms of stabilizing arteriosclerotic plaques.