Bilirubin derivative-based diagnostic and therapeutic ultrasound contrast agent

Abstract

Provided is a bilirubin derivative-based ultrasound contrast agent for diagnosis and treatment. The fine particles including the bilirubin derivative are sensitive to reactive oxygen species (ROS), bind with hydrophobic drugs, and can effectively chelate metals such as iron oxide nanoparticles. Therefore, the fine particle of the present invention can be used as an ultrasound contrast agent for diagnosis, as a magnetic resonance imaging contrast agent, or as a carrier for hydrophobic drugs or platinum-based drugs.

Claims

1. A fine particle, comprising: a core containing a gas; and a shell comprising a bilirubin derivative and surrounding the corer, wherein the bilirubin derivative is a bilirubin conjugated with a hydrophilic molecule, and the bilirubin is closer to the core than the hydrophilic molecule.

2. The fine particle of claim 1, wherein the gas is selected from the group consisting of air, nitrogen, helium, argon, carbon dioxide, sulfur hexafluoride (SF.sub.6), and C.sub.1 to C.sub.10 perfluorocarbons.

3. The fine particle of claim 1, wherein the hydrophilic molecule is selected from the group consisting of dextran, carbodextran, polysaccharide, cyclodextran, pluronic, cellulose, starch, glycogen, carbohydrate, monosaccharide, disaccharide and oligosaccharide, polypeptide, polyphosphazene, polyethylene glycol, PEG, Methoxy polyethylene glycol (methoxy polyethylene glycol, mPEG), polypropylene glycol, polyethylenimine, poly-L-lysine, polyglycolide, polymethyl methacrylate, Polyvinylpyrrolidone, poly(acrylate), poly(acrylamide), poly(vinylester), poly(vinyl alcohol) (poly[vinyl alcohol]), polystyrene, polyoxide, polyelectrolyte, poly(N-vinylpyrrolidone), poly(N-vinyl pyrrolidone), polyvinylamine, poly(beta-hydroxyethyl methacrylate), polyethylene oxide, poly(ethylene oxide-b-propylene oxide), and polylysine.

4. The fine particle of claim 1, further comprising: a metal ion or a metal compound selected from the group consisting of Cu, Ga, Rb, Zr, Y, Tc, In, Ti, Gd, Mn, Fe, Au, Pt, Zn, Na, K, Mg, Ca, Sr, and lanthanide metals.

5. The fine particle of claim 1, further comprising: an anti-cancer drug loaded in the core, wherein the anti-cancer drug is selected from the group consisting of a platinum-based anti-cancer drug, an anthracycline-based anti-cancer drug, a taxane-based anti-cancer drug, and a camptothecin-based anti-cancer drug, wherein the platinum-based anti-cancer drug is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, and heptaplatin, wherein the anthracycline-based anti-cancer drug is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, idarubicin, gemcitabine, mitoxantrone, pirarubicin, and valrubicin, wherein the taxane-based anti-cancer drug is selected from the group consisting of paclitaxel, docetaxel, and cabazitaxel.

6. The fine particle of claim 1, further comprising: a superparamagnetic iron oxide nanoparticle (SPION) which is loaded in the core.

7. An ultrasound contrast agent comprising: a core containing a gas; and a shell comprising a bilirubin derivative and surrounding the core, wherein the bilirubin derivative is a bilirubin conjugated with a hydrophilic molecule, and the bilirubin is closer to the core than the hydrophilic molecule.

8. The ultrasound contrast agent of claim 7, further comprising: a drug, wherein the ultrasound contrast agent serves as a drug delivery system.

9. A method for obtaining a diagnostic image by magnetic resonance (MR), comprising: administrating an effective amount of an ultrasound contrast agent to a subject, wherein the ultrasound contrast agent includes the particle of claim 1; and imaging a body part or a tissue of the subject by magnetic resonance (MR).

10. The method of claim 9, wherein the magnetic resonance (MR) is MR-guided focused ultrasound (MRgFUS).

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a diagram schematically illustrating a method of preparing an ultrasonic contrast agent coated with a pegylated bilirubin according to the present invention.

(2) FIG. 2 is a photograph of the ultrasonic contrast agent coated with the pegylated bilirubin according to the present invention.

(3) FIG. 3 shows representative phantom images of the ultrasonic contrast agent samples at the beginning of the measurement (t=0 min). Each of the ultrasonic contrast agent samples is coated with pegylated bilirubin. The samples had different volume ratios of perfluoropentane (PFP) in the hydrophobic gas core (PFP 0, 2.5, 5, 10% v/v).

(4) FIG. 4 is a graph showing hourly changes in phantom images of the ultrasonic contrast agent samples which are coated with pegylated bilirubin. The samples had different volume ratios of perfluoropentane (PFP) from each other in the hydrophobic gas core (PFP 0, 2.5, 5, 10% v/v).

(5) FIG. 5 is a graph showing normalized ultrasound intensity of the phantom images of the ultrasonic contrast agent samples which are coated with pegylated bilirubin over time. The samples had different volume ratios of perfluoropentane (PFP) from each other in the hydrophobic gas core (PFP 0, 2.5, 5, 10% v/v).

(6) FIG. 6 shows transmission electron microscope images of the ultrasonic contrast agent which is coated with pegylated bilirubin. The measured size of the contrast agent bubble was 2-4 μm.

(7) FIG. 7A is an image showing the ultrasonic contrast agent (white arrows) under an optical microscope. The ultrasonic contrast agent is coated with pegylated bilirubin. FIG. 7B is an image showing the ultrasound contrast agent dispensed on a hemocytometer grid. This image allowed the determination of the number of the contrast agent per volume.

(8) FIG. 8 is a graph showing a gradual increase [red(.circle-solid.), {circle around (1)}green(.square-solid.).fwdarw.{circle around (2)}blue(.box-tangle-solidup.)] in hydrodynamic size of the ultrasonic contrast agent after treatment with reactive oxygen species. The ultrasonic contrast agent is coated with pegylated bilirubin.

(9) FIG. 9 is a diagram schematically illustrating the ultrasonic contrast agent coated with PEGylated bilirubin and loaded with iron oxide nanoparticle.

(10) FIG. 10A is an image showing a position (arrow) in which PEGylated bilirubin loaded with iron oxide nanoparticles is attached to a magnet. FIG. 10B is a transmission electron microscopic image of iron oxide nanoparticle-loaded PEGylated bilirubin coated US contrast agents.

EMBODIMENTS

(11) Hereinafter, the present invention will be described in more detail with examples. It is obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention is not limited to or by the examples

EXAMPLES

Example 1: Preparation of a PEGylated Bilirubin-Based Ultrasound Contrast Agent According to the Present Invention

(12) Preparation of Bilirubin Derivative (Pegylated Bilirubin)

(13) The present inventors prepared an amphiphilic derivative of bilirubin in which a hydrophilic molecule was conjugated to bilirubin prior to preparing a bilirubin-based ultrasound contrast agent. Polyethylene glycol was used as the hydrophilic molecule.

(14) Specifically, bilirubin was first dissolved in dimethylsulfoxide (DMSO), and an appropriate amount of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) was added to activate the carboxyl group present in bilirubin, so as to induce the desired reaction. The solution was then reacted at room temperature for about 10 minutes.

(15) Next, polyethylene glycol, which has an amine group at its terminal, was added. The resulting solution is laid for reaction for a certain time period. The carboxyl group of bilirubin was covalently bonded to the amine group of polyethylene glycol via the formation of amide bonds, thereby forming the bilirubin derivative.

(16) Finally, the bilirubin derivative prepared from the above method was purified and extracted using a silica column.

(17) Preparation of Ultrasonic Contrast Agent Coated with Pegylated Bilirubin

(18) A bilirubin-based echogenic nanoparticle (or microparticle) of the present invention was prepared by a simple oil-in-water (O/W) emulsification method. A bilirubin nanoparticle particle solution (1.2 mg/2 ml) was prepared by dissolving the bilirubin derivative (Pegylated bilirubin) prepared in Example 1-1 in deionized water, and transferring the resulting solution to an ice bath equipped with a probe type ultrasonic grinder.

(19) Perfluoropentane (PFP) was used as a hydrophobic gas forming a bubble core. Perfluoropentane (organic phase) is added dropwise to the bilirubin nanoparticle particle solution (aqueous phase) at various volume ratios (PFP, 2.5, 5, 10% v/v). The resulting solution was treated with ultrasound at a power of 30% for 90 seconds. As a result, an emulsion type of nano- or micro-bubble system was prepared. The system has a core composed of hydrophobic gas (perfluoropentane) and a shell composed of bilirubin derivatives (FIGS. 1 and 2).

Example 2: Phantom Imaging of the PEGylated Bilirubin-Based Ultrasound Contrast Agent According to the Present Invention

(20) The ultrasound phantom image was obtained using an ultrasound device probe, Vevo770. The Vevo770 (High-Resolution Micro-Imaging System, Visualsonics, Toronto, Canada) is equipped with an RMV 706 probe. The present inventors used agar-gel phantoms prepared by embedding 500 μL of Eppendorf tubes in a 3% (w/v) agarose gel to simulate in vivo conditions for ultrasound imaging.

(21) First, the PEGylated bilirubin-based contrast agent samples of the present invention, each with a unique volume ratio of perfluoropentane (PFP) in the hydrophobic gas core (PFP 0, 2.5, 5, 10% v/v), was placed in agar gel phantom. The images were obtained by treating each sample with 40 MHz ultrasound.

(22) The change in the ultrasonic intensity of each sample (PFP 0, 2.5, 5, 10% v/v) was measured for 180 minutes. The measured change was normalized by subtracting the ultrasonic intensity of the water control from the sample's ultrasonic intensity. The echogenic characteristics of each sample of the PEGylated bilirubin solution, with different volume ratios of PFP in the gas core, were hourly monitored. The results are shown in FIGS. 3-5.

(23) FIG. 3 shows representative phantom images of the ultrasonic contrast agent samples at the beginning of the measurement (t=0 min). Each of the ultrasonic contrast agent samples is coated with pegylated bilirubin. The samples had different volume ratios of perfluoropentane (PFP) in the hydrophobic gas core (PFP 0, 2.5, 5, 10% v/v).

(24) FIG. 4 is a graph showing hourly changes in phantom images of the samples of the contrast agent coated with pegylated bilirubin, each with a unique volume ratio of perfluoropentane (PFP) in the hydrophobic gas core (PFP 0, 2.5, 5, 10% v/v). As shown in FIGS. 3-4, the highest level echogenicity was observed in the PFP 5.0% (v/v) experimental group.

(25) FIG. 5 is a graph showing normalized ultrasound intensity of the phantom images of the ultrasonic contrast agent samples which are coated with pegylated bilirubin over time. The samples had different volume ratios of perfluoropentane (PFP) from each other in the hydrophobic gas core (PFP 0, 2.5, 5, 10% v/v). The in-situ half-life of the echo signal of the PEGylated bilirubin-based ultrasound contrast agent was about 45 minutes.

(26) From the above results, it was confirmed that the bilirubin derivative of the present invention, which is formed by the conjugation of the hydrophilic molecule to the PEGylated bilirubin, functions as a stable shell surrounding the hydrophobic gas core. The phantom imaging also confirmed the enhancement effect on ultrasonic imaging. Therefore, the bilirubin derivative-based fine bubbles prepared according to the present invention can be useful as an ultrasound contrast agent.

Example 3: Features of the Pegylated Bilirubin-Based Ultrasound Contrast Agent According to the Present Invention

(27) 3-1. Microscope Morphology

(28) Microscopic morphology of the fine particle was observed with a negative staining of uranium acetate, a transmission electron microscope (Tecnai G2 F30, Eindhoven, Netherlands) (FIG. 6), and an optical microscope under cover slip (FIGS. 7A and 7B). FIG. 6 shows the PEGylated bilirubin-based ultrasound contrast agent of the present invention observed by transmission electron microscopy (TEM). FIG. 6 shows micro-sized bubble particles constituting the ultrasound contrast agent of the present invention.

(29) FIG. 7A shows the PEGylated bilirubin-based ultrasound contrast agent of the present invention observed with an optical microscope. In order to determine the number of bubbles contained per volume of contrast agent, the PEGylated bilirubin-based contrast agent of the present invention was placed on a hemocytometer grid and the bubbles were counted (FIG. 7B). It was confirmed that about 2.0×109 bubbles were contained per ml of the contrast agent of the present invention.

(30) 3-2. Pegylated Bilirubin-Based Ultrasound Contrast Agent Activity on Reactive Oxygen Species (ROS)

(31) The PEGylated bilirubin-based ultrasound contrast agent of the present invention includes bilirubin, which is a natural antioxidant. The present inventors used the Nanosizer ZS 90 (Malvern Instruments, Ltd., Malvern, UK) to confirm the reactivity of the ultrasonic contrast agent of the present invention to reactive oxygen species (ROS). The hydrodynamic size distribution of the microbubble of the contrast agent of the present invention before/after treatment with reactive oxygen species (ROS, H.sub.2O.sub.2) was measured. The results are shown in FIG. 8. As shown in FIG. 8, the hydrodynamic size of the bubble increased as the ultrasonic contrast agent of the present invention reacted with reactive oxygen species (H.sub.2O.sub.2).

(32) Bilirubin is a natural antioxidant in the body. When bilirubin reacts with the reactive oxygen species, which is rich in a diseased site, bilirubin is converted to biliverdin. As a result, the hydrophobic interaction between the bilirubin derivatives and the gas core is weakened, and the amphiphilic bilirubin derivative-coated shell of the contrast agent bubble is destroyed. Eventually, the instantaneous conglomeration of the hydrophobic gas core occurs. Then, the contrast of the ultrasound image is enhanced by a degree proportional to the gradual increase in size of the bubble (FIG. 8).

(33) Accordingly, the bilirubin derivatives, including the PEGylated bilirubin combined with a hydrophilic molecule, can enhance the ultrasound image of diseased sites in which the reactive oxygen species are rich. They also exhibit an antioxidant effect in the diseased sites due to their inherent antioxidant properties. Therefore, the fine particle containing the bilirubin derivative of the present invention can be useful not only for diagnosis based on ultrasound examinations but also for treatment of diseases.

Example 4: Preparation of PEGylated Bilirubin-Based Ultrasonic Contrast Agent Loaded with Iron Oxide Nanoparticle

(34) Loading of iron oxide nanoparticles was performed by modifying the method for preparation described above in Example 1. When making an oil-in-water (O/W) layer, a solution of iron oxide nanoparticles dispersed in hexane was added to the perfluoropentane (PFP) organic phase.

(35) Then, ultrasound treatment is performed to obtain an emulsion, as described above. The emulsion was agitated under dim light for 6 hours to evaporate hexane. Then, it was centrifuged at 5000 rpm and aggregates were removed. The supernatant was isolated and iron oxide nanoparticle-loaded PEGylated bilirubin microbubble was extracted using a rare earth magnet (FIG. 9 and FIGS. 10A and 10B).

(36) FIG. 9 is a diagram schematically illustrating the iron oxide nanoparticle-loaded PEGylated bilirubin-based, prepared according to the method described above. As shown in FIG. 9, in the ultrasonic contrast agent of the present invention, the hydrophobic region (hydrophilic molecule) of the bilirubin-hydrophilic polymer conjugate is exposed to the aqueous phase. The hydrophobic region (bilirubin) of the bilirubin-hydrophilic polymer conjugate is in direct contact with the hydrophobic gas (PFP) core.

(37) Here, bilirubin can bind to the iron oxide nanoparticle as follows. The oleic acid layer, coated on the iron oxide nanoparticle, falls off. Then, the carboxyl group of bilirubin binds to the iron oxide nanoparticle through a chelation reaction. Alternatively, when the core has a relatively large volume, 15 nm-sized iron oxide nanoparticles may be loaded onto the hydrophobic gas core via hydrophobic interaction.

(38) FIG. 10A shows that, when the PEGylated bilirubin contrast agent loaded with iron oxide nanoparticle is extracted with a magnet, the contrast agent containing iron oxide nanoparticle is attracted to the magnet (red arrow). FIG. 10B shows a transmission electron microscope image of an ultrasonic contrast agent coated with PEGylated bilirubin loaded with iron oxide nanoparticle. The arrows in FIG. 108 point to the iron oxide nanoparticle loaded on the microbubble of the PEGylated bilirubin-based ultrasound contrast agent according to the present invention. The size of the iron oxide nanoparticle is about 15 nm.

(39) The above results confirmed that magnetic resonance sensitive metal particles, including iron oxide nanoparticles, can be loaded to the ultrasonic contrast agent according to the present invention. The ultrasonic contrast agent includes the bilirubin derivative conjugated to the hydrophilic molecule. Therefore, the bilirubin derivative-based ultrasound contrast agent of the present invention can be used not only as an ultrasound contrast agent, but also as a contrast agent for MR-guided focused ultrasound (MRgFUS).

(40) Furthermore, the chelating properties of the metal particles of bilirubin are used to load a platinum-based anti-cancer drug, rather than iron oxide nanoparticle, on the ultrasonic contrast agent of the present invention. In this case, the contrast agent can also serve as a carrier for anti-cancer drug delivery. The magnetic resonance-guided focused ultrasound (MRgFUS) is a novel technique that can temporarily increase the permeability of the blood-brain barrier (BBB).

(41) The use of magnetic resonance-guided focused ultrasound enables the delivery of therapeutic agents into the central nervous system and increases the efficiency of the treatment of brain tumors. Therefore, the ultrasound contrast system of the present invention can be a useful platform technology capable of simultaneously performing three roles: an ultrasound contrast agent, a magnetic resonance sensitive contrast agent, and an antioxidant/anti-cancer delivery carrier.