PARTICLES COMPRISING BILIRUBIN DERIVATIVE AND METAL

Abstract

The present invention provides hydrophilic bilirubin derivative particles containing a metal, a use thereof, and a preparation method therefor. The bilirubin derivative particles of the present invention form coordinate bonds with various metals, and thus can be used in MR diagnosis, CT diagnosis, photo-acoustic diagnosis, PET diagnosis, or optical diagnosis. The bilirubin derivative particles of the present invention can release an anticancer drug encapsulated therein to the outside by the combination with a platinum-based anticancer drug and the degradation by a stimulation of light/reactive oxygen species, and exhibit anti-inflammatory and anticancer activities, and thus the bilirubin derivative particles of the present invention have a concept of theranostics in which the bilirubin derivative particles can be for therapeutic uses as well as diagnostic uses.

Claims

1. A bilirubin derivative particle comprising a bilirubin derivative and a metal.

2. The bilirubin derivative particle of claim 1, wherein the bilirubin derivative particle is configured through a coordinate bond of the bilirubin derivative and the metal,

3. The bilirubin derivative particle of claim 1, wherein the coordinate bond is formed between the metal and a carboxyl group, a lactam group, or a pyrrole ring of the bilirubin derivative.

4. The bilirubin derivative particle of claim 1, wherein the metal is an ion or compound of a metal selected from the group consisting of Cu, Ga, Rb, Zr, Y, Tc, In, Ti, Gd, Mn, Fe, Au, Pt, Pd, Ag, Co, Mn, Zn, Gd, Mo, Ni, Fe, Cr, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, Ra, and lanthanide metals.

5. The bilirubin derivative particle of claim 1, wherein the metal is a superparamagnetic iron oxide nanoparticle (SPION) or a gold nanoparticle.

6. The bilirubin derivative particle of claim 1, wherein the metal is a platinum (Pt) ion or a platinum-based anticancer drug selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, and heptaplatin.

7. The bilirubin derivative particle of claim 1, wherein the metal is a radioactive isotope selected from the group consisting of .sup.64Cu, .sup.68Ga, .sup.82Rb, .sup.89Zr, .sup.90Y, .sup.99mTc, and .sup.201Tl.

8. The bilirubin derivative particle of claim 1, wherein the bilirubin derivative is a derivative in which a hydrophilic molecule is conjugated to bilirubin.

9. The bilirubin derivative particle of claim 8, wherein the hydrophilic molecule is selected from the group consisting of dextran, carbodextran, polysaccharide, cyclodextran, pluronic, cellulose, starch, glycogen, carbohydrate, monosaccharide, bisaccharide and oligosaccharide, polyphosphagen, polylactide, poly(lactic-co-glycolic acid), polycaprolactone, polyanhydride, polymaleic acid and polymaleic acid derivatives, polyalkylcyanoacrylate, polyhydroxybutylate, polycarbonate, polyorthoester, polyethyleneglycol, polypropyleneglycol, polyethylenimine, poly-L-lysine, polyglycolide, polymetacrylate, polyvinylpyrrolidone, poly[acrylate], poly[acrylamide], poly[vinylester], poly[vinyl alcohol], polystryene, polyoxide, polyelectrolyte, poly[1-nitropropylene], poly[N-vinyl pyrrolidone], poly[vinyl amine], poly[beta-hydroxyethylmethacrylate], polyethyleneoxide, poly[ethylene oxide-bpropyleneoxide], polylysine, and peptide.

10.-15. (canceled)

16. A method for detection of reactive oxygen species, the method including the steps of: (a) contacting a suspension containing the bilirubin derivative particles of claim 1 with a sample containing reactive oxygen species; and (b) comparing and analyzing a change of the suspension before and after the contact with the sample with a control group.

17. The method of claim 16, wherein the change of the suspension in step (b) is selected from the group consisting of the presence or absence of precipitation of bilirubin derivative particles, the absorbance according to wavelength, the transparency of the suspension, the concentration of metal ions in the suspension, and the intensity of MRI image signal.

18. A method comprising a step of administering, to a subject, a composition comprising the bilirubin derivative particle of claim 1.

19. The method of claim 18, wherein the method is for image diagnosis.

20. The method of claim 18, wherein the method is for treatment of cancer.

21. The method of claim 18, wherein the method is for treatment and diagnosis of an inflammatory disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0156] FIG. 1 shows examples of application using bilirubin derivative particles of the present invention.

[0157] FIG. 2 shows a preparation procedure for bilirubin derivative particles of the present invention and a labeling procedure using the radioactive isotope .sup.64Cu for the use of PET imaging.

[0158] FIG. 3 shows labeling efficiency according to pH and temperature in order to investigate reaction conditions for optimizing radioactive labeling efficiency.

[0159] FIG. 4 shows representative micro-PET images of PC-3 tumor (yellow arrows)-retaining mice 1, 3, and 6 hr after intravenous injection of .sup.64Cu-bilirubin particles (axial images, upper panels; and coronal images, lower panels).

[0160] FIG. 5a illustrates the colorimetric measurement in the reaction of PEG-bilirubin and metal ions and provides images of a suspension of bilirubin particles before (upper panel) and after (lower panel) the reaction with particular metal ions.

[0161] FIG. 5b illustrates the colorimetric measurement in the reaction of PEG-bilirubin and metal ions and shows UV/Vis spectra for a suspension of bilirubin particles before and after the reaction with particular metal ions.

[0162] FIG. 6a shows the preparation of an iron oxide-based MR probe using PEG-bilirubin. (1) In the lipid film method on the left side, the bilirubin derivative particles having a metal encapsulated therein of the present invention are produced such that clustered iron oxide is placed at the center and the PEG-bilirubin layer surrounds the iron oxide. (2) In the sonication method on the right side, uniform iron oxide nanoparticles coated with the PEG-bilirubin layer are produced.

[0163] FIG. 6b shows the principle of coordinate bonding of PEG-bilirubin and superparamagnetic iron oxide.

[0164] FIG. 6c provides representative TEM images showing clustered iron oxide nanoparticles with a PEG-bilirubin shell (left side) and uniformly distributed iron oxide nanoparticles with PEG-bilirubin shells (right side).

[0165] FIG. 7 shows the features of PEG-bilirubin coated SPIONs and provides the T2-weighted MR phantom images of PEG-bilirubin coated SPIONs in an aqueous solution and the T2 relaxation rate as a function of ion concentration.

[0166] FIG. 8 shows the features of PEG-bilirubin coated SPIONs and provides TEM images of PEG-bilirubin coated SPIONs before and after ROS stimulation.

[0167] FIG. 9a shows the features of PEG-bilirubin coated SPIONs and provides sequential MR phantom images of ROS-responsiveness according to the ROS concentration, of PEG-bilirubin coated SPIONs.

[0168] FIG. 9b shows the features of PEG-bilirubin coated SPIONs and provides a graph of T2 relaxation value change according to ROS concentration in PEG-bilirubin coated SPIONs.

[0169] FIG. 10 shows the intra-macrophage expression level of Nox2 gene, measured by RT qPCR.

[0170] FIG. 11a shows the observation results, through an optical microscope, of the level of macrophage phagocytosis in PEG-DSPE coated SPION and PEG-BR SPION treatment groups under ROS production conditions.

[0171] FIG. 11b shows the comparison, through MRI phantom experiment, of the level of macrophage phagocytosis in PEG-DSPE coated SPION and PEG-BR SPION treatment groups targeting the macrophages collected from the mouse peritoneal cavity.

[0172] FIG. 12 shows that when the PEG-BR coated gold nanoparticles react with reactive oxygen species, the PEG-BR coating was peeled off, resulting in the aggregation of gold nanoparticles, thereby producing a potent photothermal effect in a near-infrared (NIR) region.

[0173] FIG. 13 shows the CT imaging results of mice using PEG-BR coated gold nanoparticles.

[0174] FIG. 14 shows a negatively stained TEM image of cisplatin-loaded bilirubin particles.

[0175] FIG. 15 shows cisplatin chelation and provides an image of suspensions (left side) and a graph of UV/Vis spectra (right side) of general bilirubin particles (BRNP) and bilirubin particles reacting with cisplatin (BRNP+Cisplatin).

[0176] FIG. 16 shows an estimated reaction mechanism of a PEG-bilirubin particle and cisplatin.

[0177] FIG. 17a and FIG. 17b show cisplatin release patterns according to several conditions (pH and ROS) and time in cisplatin-encapsulated PEG-bilirubin particles.

[0178] FIG. 18 shows in vivo photo-acoustic images over time after injection into xenograft tumor of a nude mouse and semi-quantitative analysis of pixel values in the tumor corresponding thereto.

[0179] FIG. 19 shows infrared thermal images at different time intervals of a tumor xenograft mouse exposed to a near infrared (NIR) laser at an output power of 800 mW/cm.sup.2.

[0180] FIG. 20 and FIG. 21 show the results of observation according to the period when cisplatin-encapsulated PEG-bilirubin particles were injected into a xenograft tumor of a nude mouse and then a photothermal therapy was performed on the nude mouse using light.

[0181] FIG. 22 shows the change of an aqueous solution of PEGylated bilirubin coated iron nanoparticles of the present invention according to the concentration of reactive oxygen species.

[0182] FIG. 23 shows the change of an aqueous solution of PEGylated bilirubin-coated iron nanoparticles of the present invention according to the concentration of NaOCl as reactive oxygen species.

[0183] FIG. 24 shows the change of an aqueous solution of PEGylated bilirubin coated iron nanoparticles of the present invention according to the concentration of 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) as reactive oxygen species.

[0184] FIG. 25 shows the change of an aqueous solution of PEGylated bilirubin coated iron nanoparticles of the present invention according to the concentration of hydrogen peroxide water as reactive oxygen species.

[0185] FIG. 26 shows the visible changes of the aqueous solution of PEGylated bilirubin-coated gold nanoparticles (PEG-BR GNP) of the present invention before and after reaction with respective types of reactive oxygen species (H.sub.2O.sub.2, NaOCl, and AAPH).

[0186] FIG. 27 shows the absorbance changes of the aqueous solution of PEGylated bilirubin coated gold nanoparticles (PEG-bilirubin gold nanoparticle) of the present invention before and after reaction with respective types of reactive oxygen species (H.sub.2O.sub.2, NaOCl, AAPH).

[0187] FIG. 28 shows the absorbance changes of the aqueous solution of PEG-thiol coated gold nanoparticles, as a control group for the PEGylated bilirubin of the present invention, before and after reaction with respective types of reactive oxygen species (H.sub.2O.sub.2, NaOCl, and AAPH).

[0188] FIG. 29 is a schematic diagram of a bilirubin derivative particle prepared by the manganese ion (Mn2.sup.+) coordination.

[0189] FIG. 30 shows a preparation process for bilirubin derivative nanoparticles coordinating manganese ion (Mn.sup.2+) as a paramagnetic element for use in MRI imaging. PEG-bilirubin is used to form nanoparticles, which are then mixed with manganese ions, to thereby producing manganese ion-coordinated particles.

[0190] FIG. 31 is a schematic diagram showing that the particles containing a bilirubin derivative and a metal, of the present invention, can detect or diagnose reactive oxygen species. Specifically, when the manganese ion-coordinated bilirubin derivative reacts with reactive oxygen species, hydrophobic bilirubin was transformed into hydrophilic biliverdin or degraded into bilirubin fragments, leading to weakened binding and nanoparticle breakdown. As a result, the coordinated manganese ions were separated, leading to MRI image enhancement.

[0191] FIG. 32 shows a pattern in which the manganese ion-coordinated bilirubin derivative particles release manganese ions by the stimulation of reactive oxygen species.

[0192] FIG. 33 shows TEM images before and after the manganese ion-coordinated bilirubin derivative (PEG-BR) nanoparticles were treated with hypochlorite as a reactive oxygen generator.

[0193] FIG. 34 shows MRI T1 weighted images before and after the treatment of manganese-coordinated bilirubin nanoparticle with reactive oxygen species (hypochlorite).

DETAILED DESCRIPTION

[0194] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLES

Example 1: Preparation of Bilirubin Derivative (PEG-BR) Particles of the Present Invention

[0195] 1-1. Preparation of Bilirubin Derivative (PEG-BR)

[0196] The present inventors prepared a bilirubin derivative in which polyethylene glycol as a hydrophilic molecule is conjugated to bilirubin, as a previous step for preparing a bilirubin derivative particles containing bilirubin and a metal, using a complexation effect of bilirubin.

[0197] First, bilirubin was dissolved in dimethyl sulfoxide (DMSO), and then, in order to activate a carboxyl group present in bilirubin to induce a desired reaction, an appropriate amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added thereto, followed by reaction at room temperature for 10 minutes. Then, polyethylene glycol having an amine group at an end thereof was added thereto, followed by reaction for a predetermined period of time, thereby synthesizing a bilirubin derivative in which a carboxyl group of bilirubin is conjugated to an amine group of polyethylene glycol through an amide bond. Last, the finally prepared bilirubin derivative was purely isolated and extracted from byproducts through a silica column.

[0198] 1-2. Preparation of Bilirubin Derivative (PEG-BR) Particles

[0199] The polyethylene glycol-conjugated amphiphilic bilirubin derivative, which was prepared in example 1-1 above, was dissolved in an organic solvent, such as chloroform or dimethyl sulfoxide, followed by drying under nitrogen gas conditions, to thereby form a lipid film layer. The prepared lipid film layer of bilirubin derivative was hydrated with an aqueous solution to prepare self-assembled bilirubin particles dissolved in the aqueous solution.

Example 2: Preparation of Bilirubin Derivative Particles Containing Metal (Metal Ion) of the Present Invention1

[0200] 2-1. Preparation of Bilirubin Derivative Particles Containing .sup.64Cu Ion as Radioactive Isotope and In Vivo PET Imaging Thereof

[0201] In order to investigate the metal ion encapsulation effect of the bilirubin derivative particles of the present invention, the following experiment was conducted. An aqueous solution of a small amount of radioactive isotope .sup.64CuCl.sub.2, which is used in PET image diagnosis, was mixed with an aqueous solution of the bilirubin derivative particles prepared in example 1 without a separate additive. Then, a reaction occurred very intensively and rapidly to load .sup.64Cu ions with only a reaction time of about 30 minutes (FIG. 2).

[0202] In addition, in order to investigate how great active the bilirubin derivative particles of the present invention are, the free .sup.64Cu not contained in bilirubin was removed using a size exclusion column, and then quantified with a radiation dosimeter. Meanwhile, the reaction methods under other pH and temperature conditions in the 64Cu chelation were optimized to be almost identical to the physiological environment (37 C., pH 7.4) (FIG. 3).

[0203] The coordinate bond between the .sup.64Cu ion and the bilirubin derivative of the present invention may be formed by the .sup.64Cu ion and a pyrrole group, lactam group, or carboxyl group of bilirubin, and an exemplary expression thereof is shown in chemical formula 2.

##STR00002##

[0204] In addition, the bilirubin derivative particles, in which the .sup.64Cu ion is coordinated by the bilirubin derivative (PEG-BR) particles prepared in example 1 above, were injected into rats with a tumor, and in vivo performance thereof was preliminarily investigated by PET imaging. As a confirmation results, the .sup.64Cu-bilirubin particles clearly visualized the tumor in a time-dependent manner, and the highest tumor uptakes at 1 h, 3 h and 6 h after injection were about 2.15, 2.81, and 3.75% injected dose (ID)/g, respectively (FIG. 4).

[0205] 2-2. Preparation of Bilirubin Derivative Particles Containing Various Metal (Ni, Mn, Gd, Mg, Ca, Fe) Ions

[0206] In order to investigate the encapsulation effect (chelating effect) of the bilirubin derivative particles of the present invention with respect to various metal ions, the possibility of coordination complex formation for six metals (Ni, Mn, Gd, Mg, Ca, and Fe) was examined. As for an experimental method, an aqueous solution containing each of the metal ions was added to an aqueous solution of the bilirubin particles prepared in example 1, followed by mixing, as in example 2-1 above. After a certain reaction time, all the metals, especially transition metals, exhibited color changes (NiFe>GdMn), which were distinctive when compared with the color changes of Mg and Ca (FIG. 5a). In addition, the respective metals, even magnesium and calcium groups, showed various absorbance pattern changes compared with general particle solutions (FIG. 5b).

[0207] The above results that, after forming coordinate bonding with particular metal ions, the bilirubin particle solution showed color changes from the original yellow color thereof or showed a displacement or change in particular absorbance pattern, may provide the applicability of novel PEGylated bilirubin beyond previous biomedical application fields Therefore, it could be confirmed that the ability of the bilirubin derivate particles of the present invention to form a metal-organic coordination complex for various metals can be used for various applications including a metal ion detection system.

Example 3: Preparation of Bilirubin Derivative Particles Containing Metal (Metal Nanoparticles) of the Present Invention2

[0208] 3-1. Preparation of Bilirubin Derivative Particles Containing Single Superparamagnetic Iron Oxide Nanoparticle (SPION)

[0209] In order to coat superparamagnetic iron oxide nanoparticles (SPIONs) with the bilirubin derivative of the present invention, a hexane solution containing superparamagnetic iron oxide nanoparticles (SPIONs) dissolved therein was added to the aqueous solution of the bilirubin nanoparticles prepared in example 1 above, to thereby form an interface portion through the separation of a water layer and a hexane layer. The artificial pressure is applied to the interface portion using a sonicator to mix the two layers for a predetermined period of time, thereby preparing particles in the form in which the bilirubin derivative (PEG-BR) is coated on surfaces of the iron nanoparticles (FIG. 6a). The above reaction is on the basis of a principle of ligand exchange, in which an oleic acid layer, which is originally coated on the iron nanoparticle (SPIONs), is separated, and instead, through a chelation reaction of the carboxyl group of the bilirubin derivative (PEG-BR) and a core portion of the iron nanoparticles (SPIONs), metal nanoparticles were coated (FIG. 6b).

[0210] 3-2. Preparation of Bilirubin Derivative Particles Containing Clustered Form of Superparamagnetic Iron Oxide Nanoparticles (SPIONs)

[0211] In order to prepare particles in the form in which metal particle clusters are coated with a bilirubin derivative, SPION particles dissolved in methanol were mixed with bilirubin derivative (PEG-RB) dissolved in an organic solvent (e.g., chloroform), instead of adding metal particles dissolved in an organic solvent to an aqueous solution containing a bilirubin derivative (PEG-BR) dissolved therein as in example 3-1. Thereafter, the organic solvent was dried under nitrogen gas conditions to form a lipid film layer. Last, the formed lipid film layer was hydrated to form a cluster form of SPIONs. Pure SPIONs were isolated through a magnet after passing through the above procedures, and a cluster form of SPIONs prepared thereby was isolated as a final reaction product.

[0212] The present inventors confirmed from TEM images that two types of particles in examples 3-1 and 3-2 were successfully prepared using PEG-bilirubin shells (FIG. 6c).

[0213] 3-3. Preparation of Bilirubin Derivative Particles Containing Gold Nanoparticle

[0214] In order to coat gold nanoparticles with a bilirubin derivative, the bilirubin derivative (PEG-BR) prepared in example 1-1 above was dissolved in water rather than an organic solvent, followed by immediate reaction with an aqueous solution containing gold nanoparticles dissolved therein for a predetermined period of time. The principle of the reaction is similar to the principle of SPION coating in example 3-1. The bilirubin derivative (PEG-BR), in substitution for a citrate layer, coated on the gold nanoparticles, was coated while surrounding a nanoparticle core.

Example 4: ROS-Responsiveness of Bilirubin Derivative Particles Containing Metal (Metal Nanoparticles) of the Present Invention

[0215] 4-1. MRI Phantom Study of Bilirubin Derivative Particles Containing SPION

[0216] The present inventors conducted the MRI phantom study in order to study characteristics of SPION coated with PEG-bilirubin in the form of mono-distribution particles.

[0217] First, as a result of comparing phantom images of the bilirubin derivative (PEG-BR) coated SPIONs of the present invention and Feridex, which is a clinically approved T2-weighted MR agent, the bilirubin derivative (PEG-BR) coated SPIONs of the present invention showed a more excellent relaxivity value than Feridex (FIG. 7).

[0218] In addition, when the bilirubin derivative (PEG-bilirubin) coated SPIONs were treated with hypochlorite as an ROX generator, the aggregation of PEG-bilirubin coated SPIONs was observed from the TEM image due to ROS-responsiveness inherent to bilirubin (FIG. 8). In addition, as predicted, the ROS-responsiveness by a gradual reduction of T2 signal, which is proportional to ROS concentration, due to the loss of hydrophilicity maintained by PEG-bilirubin was also indirectly validated in T2 MR phantom studies (FIGS. 9a and 9b). Such a SPION aggregation response can gradually increase the size of SPION, and thus can be a potent target for the therapeutic effect of magnetic hyperthermia.

[0219] 4-2. In Vitro and In Vivo Tests for ROS-Responsiveness of Bilirubin Derivative Particles Containing SPION

[0220] In order to investigate whether the PEG-bilirubin coated iron nanoparticles act and aggregate in response to ROS in vitro and in vivo, the following experiment was conducted using primary macrophages and macrophage strains, which are well known to phagocytize foreign pathogens through ROS and phagocytosis in inflammation conditions.

[0221] First, macrophages or peritoneal cavity was treated with lipopolysaccharides (LPS) to make an artificial inflammation condition, and then at the same time, subjected to treatment with PEG-bilirubin coated SPIONs and PEG-distearoylphosphatidylethanolamine (PEG-DSPE) coated SPIONs as a control group, and the phagocytosis patterns thereof were observed using an optical microscope and an MR phantom images.

[0222] In order to investigate whether ROS was produced in the same amount in respective conditions, the intra-macrophage expression level of Nox2 gene, known as an ROS generation factor in the body, was measured by RT_qPCR. As a result, the PEG-DSPE and PEG-RB SPION groups showed almost similar expression levels of Nox2 gene, and produced similar amounts of ROS, which were higher compared with normal conditions (FIG. 10).

[0223] In addition, the respective phagocytosis degrees of the PEG-DSPE coated SPION and PEG-BR coated SPION treatment groups in conditions of generating an equivalent amount of ROS were observed through an optoelectronic device. As a result of observation, the PEG-BR coated SPION treatment group showed higher phagocytosis levels than PEG-DSPE coated SPION treatment group as a control group (FIG. 11a, darker color being observed with increasing degree of phagocytosis). The MRI cell phantom studies for macrophages taken from the peritoneal cavity also showed the same pattern (FIG. 11b).

[0224] The above results are thought to result from the fact that the PEG-BR coating was peeled off from the PEG-BR coated SPION in response to ROS generated from the macrophage with stress increased by LPS treatment, so that the SPION cores aggregates, leading to increased phagocytosis, or the PEG-BR coated SPIONs also aggregate in cells in response to ROS after phagocytosis. Whereas, the PEG-DSPE coated SPIONs as a control group did not perform any reaction with ROS, and thus the activity of a relatively complete form of PEG-DSPE coated SPIONs per se is thought to be observed.

[0225] 4-3. CT Phantom Studies and In Vitro and In Vivo Tests for ROS-Responsiveness of Bilirubin Derivative Particles Containing Gold Nanoparticle

[0226] Gold nanoparticles have been widely studied as a CT contrast agent in a preclinical field. A surface of the gold nanoparticle coated with citric acid, like SPION, may be substituted with PEGylated bilirubin (PEG-BR) by coordinate boning. The successful binding of the PEGylated bilirubin to the gold nanoparticle was confirmed through TEM images and CT phantom images, and the UV-Vis wavelength change after chelation and ROS-responsiveness of the gold nanoparticle coated with bilirubin was investigated by comparing and observing the gold nanoparticle coated with PEGylated thiol as a control group.

[0227] In addition, when the PEG-BR coated gold nanoparticles react with ROS, the PEG-BR coating was peeled off, resulting in the aggregation of gold nanoparticles with a loss of ligands, so that the gold nanoparticles had a potent photothermal effect in an NIR region, leading to a change in absorbance (FIG. 12). This indicates a possibility that a contrast agent based on PEG-BR coated gold nanoparticles can be used not only as a diagnostic tool using CT, but also as a tool for promoting the treatment by photothermal effects in response to ROS in tumors.

[0228] In addition, as a result of investigating the CT images of the PEGylated bilirubin coated gold nanoparticles in mice in vivo, it was confirmed that angiography can be performed with long circulation for a long period of time and excellent results were also obtained in imaging major organs, such as liver and spleen (FIG. 13).

Example 5: Preparation of Bilirubin Derivative Particles Containing Metal (Platinum-Based Anticancer Drug) of the Present Invention-3

[0229] In order to validate the chelating effect of forming a complex with a metal and the therapeutic efficacy against a tumor, of the bilirubin derivative particles of the present invention, nanoparticles loading cisplatin, which is the most representative metal drug used for a tumor, were fabricated (FIG. 14). Cisplatin has a platinum metal backbone, and has been used together with a nano-delivery system.

[0230] As a result of reaction of PEGylated bilirubin (PEG-BR) particles and a hydrolysis product of cisplatin, it was confirmed that cisplatin was loaded with an unprecedented color change in the solution (FIG. 15). A schematic diagram showing the binding principle between the PEGylated bilirubin and cisplatin is shown in FIG. 16.

[0231] In addition, as a result of conducting a cisplatin release test according to several conditions (pH and ROS) and time in cisplatin-encapsulated bilirubin nanoparticles, cisplatin showed the highest release rate in response to ROS, to show the highest release proportion, followed by a high release rate at acidic conditions (pH 5.5), which was known to be similar to environment of intracellular lysosomes, and the lowest release at physiological pH (FIGS. 17a and 17b).

[0232] The above results indirectly confirmed that the bilirubin derivative particle containing a metal of the present invention can stably encapsulate cisplatin as a platinum-based drug therein and selectively release the encapsulated drug to the surrounding micro-environment.

Example 6: Photo-Acoustic and Photothermal Activities of Bilirubin Derivative Particles Containing Metal (Platinum-Based Anticancer Drug) of the Present Invention

[0233] In the sacrifice of the decreased Soret band peak, the increase in absorbance at the IR region (red shift) induces remarkable photothermal activity at a light of 808 nm. Since the bilirubin nanoparticle per se has remarkable IR light sensitivity, the photothermal activity could not be derived from an original general IR light source. Such a change and newly acquired photon characteristics can be explained by the Platinum Blues theory. According to the theory, the hydrolysis product of cisplatin can be obtained from the reaction with an amide ligand.

[0234] As for the nanoparticles of the present invention in which PEGylated bilirubin is coordinated to cisplatin, the present inventors used such a metal-coordination complex for photo-acoustic imaging and photo-thermal therapy (PTT) due to the newly obtained absorbance in the near infrared region (NIR region). The photo-acoustic imaging and photothermal therapy share the same principle in light of a particular wavelength.

[0235] Upon application to in vivo photo-acoustical imaging in tumor xenograft model mice, it was confirmed that the photo-acoustic signal was gradually increased after the intravenous injection of the bilirubin derivative of the present invention (FIG. 18). Therefore, the possibility of photo-thermal therapy was confirmed in the same conditions, and the surface temperature of a tumor was rapidly increased to 55-60 C. within 5 minutes after exposure to light of 808 nm (FIG. 19). Resultingly, a significant tumor volume reduction effect over time was observed in the group subjected to photo-thermal therapy using actual light (FIGS. 20 and 21).

Example 7: ROS-Responsiveness of Bilirubin Derivative Particles Containing Metal of the Present Invention

[0236] 7-1. Confirmation of ROS-Responsiveness of Bilirubin Derivative Particles Containing Iron Nanoparticles (Visible Color Change)

[0237] In order to investigate ROS-responsiveness of the bilirubin derivative particles of the present invention and a change thereof, the change of PEGylated bilirubin coated iron nanoparticles of the present invention according to the concentration of ROS was examined.

[0238] First, a suspension containing PEGylated bilirubin coated iron nanoparticles was prepared by the same method as in the above-described example, and NaOCl (100, 10, 1, 0.1, 0 mM) and AAPH* (100, 10, 1, 0.1, 0 mM), and hydrogen peroxide water (100 mM) were added according to the concentration, and the results were observed with the naked eye and by an optical microscope. [*2,2-Azobis(2-am idinopropane) dihydrochloride (AAPH)]. In addition, PEGylated DSPE** coated iron nanoparticles as a negative control were used. [**1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)]

[0239] The results are shown in FIGS. 22 to 25.

[0240] As shown in FIG. 22, at a concentration of NaOCl of 100 mM, all the PEGylated bilirubin, which has coated the iron nanoparticles, were dropped due to a high concentration of ROS, and thus the remaining hydrophobic iron nanoparticles aggregate each other and settled down. As a result, the inherent color of the iron nanoparticle aqueous solution seen in the right two tubes was also lost, thus giving a clear water color. Whereas only a very small amount aggregated (red arrow) in the middle tube group treated with 1 mM as an intermediate concentration, and showed a darker coffee color due to weaker aggregation of iron nanoparticles compared with a control group (1 mM) on the right side.

[0241] As shown in FIGS. 23 to 25, it was confirmed that the ROS-responsiveness was different in the order of hypochlorite (HOCl)>>AAPH>>>>>hydrogen peroxide water. It was also confirmed that PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) coated iron nanoparticles, used as a negative control, did not react with any of three types of ROS. Therefore, it could be confirmed that the reaction of the ROS and the PEGylated bilirubin coated iron nanoparticles was very specific.

[0242] 7-2. Confirmation of ROS-Responsiveness of Bilirubin Derivative Particles Containing Gold Nanoparticle (Absorbance Change)

[0243] In order to quantitatively investigate ROS-responsiveness of the bilirubin derivative particles of the present invention, the absorbance before and after the reaction of the bilirubin derivative particles and ROS was measured. Specifically, the change of the solution before and after the reaction of PEGylated bilirubin coated gold nanoparticles with each type of ROS was measured using naked eyes and absorbance.

[0244] The results are shown in FIGS. 26 and 28.

[0245] As for AAPH, only PEGylated bilirubin coated gold nanoparticles specifically reacted with AAPH, and PEGylated thiol (PEG-SH) coated gold nanoparticles, used as a negative control, did not react with AAPH. As for hypochlorite (HOCI), both of PEGylated bilirubin coated gold nanoparticles and PEGylated thiol (PEG-SH) coated gold nanoparticles were observed to react with hypochlorite, and thus it was confirmed that the bilirubin derivative particles of the present invention showed higher response specifically to ROS (AAPH).

[0246] It could be seen from the above results that the bilirubin derivative particles of the present invention can be favorably used in determining the type and concentration of ROS.

Example 8: ROS-Responsiveness of Manganese Ion-Coordinated Bilirubin Derivative Particles of the Present Invention

[0247] 8-1. Preparation of Manganese Ion-Coordinated Bilirubin Derivative Particles

[0248] In order to further investigate the ROS-responsive and change of the bilirubin derivative particles containing a metal of the present invention, bilirubin derivative particles were prepared by the manganese ion (Mn2+) coordination. A schematic diagram and a preparation method of the manganese ion-coordinated bilirubin derivative particles are shown in FIGS. 29 and 30. Specifically, a MnCl.sub.2 aqueous solution was dropped using a syringe pump such that the molar ratio of PEG-BR:MnCl.sub.2 was 1:1 while strongly mixing an aqueous solution of the bilirubin derivative (PEG-BR) particles prepared in example 1 above (step 5)). Thereafter, a reaction was performed at 37 C. for 48 hours. After the reaction was completed, the manganese ions not bound with the bilirubin nanoparticles were removed by using a dialysis bag (float A-Lyzer, MW cutoff: 20K) (step 6)), followed by concentration using Amicon 10K (step 7)), thereby preparing manganese ion-coordinated bilirubin derivative nanoparticles. In order to measure the amount of manganese ions bound to the prepared bilirubin derivative nanoparticles, ICP-OES (Agilent ICP-OES 5110) was used (step 8)). As a result, it was confirmed that 22.672.20 mg/kg (based on PEG-BR 1 mM) of manganese ions were bound in the manganese ion-coordinated bilirubin derivative nanoparticles of the present invention.

[0249] 8-2. Confirmation of ROS-Responsiveness of Manganese Ion-Coordinated Bilirubin Derivative Particles (Ion Concentration, TEM Imaging, and MR Imaging)

[0250] In order to investigate the reaction of the manganese ion-coordinated bilirubin derivative particles of the present invention, prepared in example 8-1, with ROS, hypochlorite was added to the manganese ion-coordinated bilirubin derivate particles to obtain the release amount of manganese ions and MRI T1 weighted images therefor. FIG. 31 illustrates that when the manganese ion-coordinated bilirubin derivative of the present invention reacted with ROS, the hydrophilic bilirubin was transformed into hydrophilic biliverdin, leading to weaken binding and nanoparticle breakdown, and as a result, the coordinated manganese ions were separated, and thus the ROS were imaged using MRI.

[0251] Specifically, 1 ml of the manganese ion-coordinated bilirubin nanoparticles were added into a dialysis bag (Float A-Lyzer, MW cutoff: 20K), and 1 mM NaOCl was added to 99 ml of distilled water, and then the dialysis of manganese ions separated from the coordination state was carried out at room temperature with shaking. At predetermined times (0, 1, 2, 3, 6, 12, 24, 48 and 72 hr), 50 ul of fractions were collected from the inside of the dialysis bag, and the amount of manganese contained in each fraction was determined through ICP-MS (Agilent ICP-MS 7700S).

[0252] The results are shown FIG. 32. As shown in FIG. 32, the manganese ion-coordinated bilirubin derivative particles of the present invention released manganese ions by ROS stimulation.

[0253] The present inventors also observed, through a transmission electron microscope, the morphological changes of the manganese ion-coordinated bilirubin derivative (PEG-BR) nanoparticles of the present invention before and after the treatment with hypochlorite. The results are shown FIG. 33. As shown in FIG. 33, it could be confirmed that the manganese ion-coordinated bilirubin derivative particles gathered together at one place to form a small sphere before the stimulation of ROS (hypochlorite), but after the stimulation, the particles did not gather but are dispersed since the binding of manganese ion and bilirubin was transformed.

[0254] The present inventors also observed MR image signal intensity changes of the manganese ion-coordinated bilirubin derivative (PEG-BR) nanoparticles of the present invention before and after the treatment with hypochlorite. A 3-Tesla MRS 3000 scanner (w/a birdcage rat head coil, MR Solutions, Surrey, United Kingdom) with a 17-cm bore size was used as a measuring instrument, and the measurement parameters of the horizontal T1-weighted images were as follows:

[0255] Time of repetition (TR)/echo time (TE); 550 ms/11 ms, flip angle; 90, field of view (FOV); 45 mm45 mm, slice thickness; 1.5 mm, matrix number; 256128.

[0256] The results are shown in FIG. 1 and table 1.

TABLE-US-00001 TABLE 1 Signal-to-noise ratio (T/N contrast ratio) = (mean signal intensity)/{(standard deviation of noise intensity) * 100} Before NaOCl 10003/(110*100) = 90.9% treatment After NaOCl 19024/(110*100) = 172.9% treatment

[0257] As shown in Table 1 and FIG. 34, it could be confirmed that the manganese ion-coordinated bilirubin derivative (PEG-BR) nanoparticles of the present invention showed enhanced brightness of the MRI T1 weighted image after the treatment with hypochlorite.

[0258] Therefore, it was confirmed from the above results that the bilirubin derivative particles of the present invention can be favorably used as a composition for detection of ROS or an inflammation site accompanied by the ROS.

[0259] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention.