Metal oxide nanoparticle-based T1-T2 dual-mode magnetic resonance imaging contrast agent

Abstract

The present invention relates to a magnetic resonance imaging (MRI) contrast agent, particularly a metal oxide nanoparticle-based T1-T2 dual-mode MRI contrast agent that can be used not only as a T1 MRI contrast agent but also as a T2 MRI contrast agent, and a method for producing the same. The metal oxide nanoparticle-based T1-T2 dual-mode MRI contrast agent can provide more accurate and detailed information associated with disease than single MRI contrast agent by the beneficial contrast effects in both T1 imaging with high tissue resolution and T2 imaging with high feasibility on detection of a lesion.

Claims

1. A method for producing a T1-T2 dual-mode MRI contrast agent derived from nanoparticles that have a core of manganese oxide and a porous shell of manganese ion-doped iron oxide on the core, comprising the following steps: A) synthesizing manganese oxide nanoparticles under argon gas environment; B) forming an epitaxial layer of iron oxide on the surface of manganese oxide nanoparticles under argon gas environment; C) maintaining the formation of the layer of porous manganese ion-doped iron oxide under dry air environment to form multilayer nanoparticles having a porous shell adjacent to core structure; and D) coating multilayer nanoparticles with a pyrenyl polyethylene glycol, wherein the manganese oxide is octahedral shape or cross shape.

2. The method for producing a T1-T2 dual-mode MRI contrast agent according to claim 1, wherein the pyrenyl polyethylene glycol is modified by conjugation with targeting moieties or diagnostic moieties.

3. The method for producing a T1-T2 dual-mode MRI contrast agent according to claim 2, wherein the targeting moiety is selected from the group consisting of antibodies, antibody fragments, aptamers, and various ligands binding to receptors displayed on the surface of target cell.

4. The method for producing a T1-T2 dual-mode MRI contrast agent according to claim 2, wherein the diagnostic moiety is selected from the group consisting of fluorophores, optical reporters, quantum dots, computed tomography probes and nonmetallic radioisotopes.

Description

DESCRIPTION OF DRAWINGS

(1) The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

(2) FIG. 1 is a sectional view illustrating one exemplary type of MRI contrast agent of the present invention.

(3) FIG. 2 is a transmission electron microscopy (TEM) image of octahedral manganese (II) oxide (MnO) nanoparticles. (a) Low magnification, and (b) high magnification.

(4) FIG. 3 is an X-ray diffraction (XRD) pattern of octahedral MnO nanoparticles as synthesized.

(5) FIG. 4 is a TEM image of cross-shaped MnO nanoparticles.

(6) FIG. 5 is a TEM image of urchin-shaped MnO nanoparticles.

(7) FIG. 6 is a TEM image of cubic MnO nanoparticles.

(8) FIG. 7 is a diagram illustrating the synthesis scheme of nanoparticles of the present invention. (a) MnO nanoparticle and (b) iron oxide nanoparticle with a central MnO phase.

(9) FIG. 8 is a TEM image of the iron oxide nanoparticle with a central MnO phase derived from octahedral MnO nanoparticles according to the present invention. (a) Low magnification, and (b) high magnification.

(10) FIG. 9 is an XRD pattern of the iron oxide nanoparticle with a central MnO phase derived from octahedral MnO nanoparticles according to the present invention.

(11) FIG. 10 is a result of electron energy loss spectroscopy (EELS) analysis of the iron oxide nanoparticle with a central MnO phase derived from octahedral MnO nanoparticles according to the present invention, showing the elemental mapping images for oxygen (white), iron (red) and manganese (green).

(12) FIG. 11 is a TEM image of the iron oxide nanoparticle with a central MnO phase derived from cross-shaped MnO nanoparticles according to the present invention.

(13) FIG. 12 is a TEM image of the iron oxide nanoparticle with a central MnO phase derived from urchin-shaped MnO nanoparticles according to the present invention.

(14) FIG. 13 is a TEM image of the iron oxide nanoparticle with a central MnO phase derived from cubic MnO nanoparticles according to the present invention.

(15) FIG. 14 is a T2-weighted MR image obtained using the MRI contrast agents of the present invention.

(16) FIG. 15 is a T1-weighted MR image obtained using the MRI contrast agents of the present invention.

MODE FOR INVENTION

(17) As explained hereinbefore, the present invention is to provide a metal oxide nanoparticle-based T1-T2 dual-mode MRI contrast agent that can be used not only as a T1 MRI contrast agent but also as a T2 MRI contrast agent and a method for producing the same.

(18) Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

(19) However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Preparation of Manganese (II) Oxide Nanoparticles with Various Shapes

(20) <1-1> Preparation of Octahedral Manganese Oxide Nanoparticles

(21) The octahedral manganese (II) oxide nanoparticles were synthesized by using the method reported (Chem. Mater. 18: 1821, 2006) with some modifications. Briefly, manganese (II) formate (Mn(HCOO).sub.2, 5 mmol), oleic acid (13 mmol) and trioctylamine (15 mmol) were mixed in a 50 ml round-bottom flask. The mixture was heated in an oil bath to 120° C. with a magnetic stirring and kept at that temperature for 3 hours under a strong flow of argon gas. Then the temperature was increased to 330° C. with the heating rate of 30° C. per minute and the reaction was kept at that temperature until the green color appeared. The green solids were obtained by cooling the reaction solution down to room temperature and were washed with 1-propanol followed by a centrifugation (3 min, 3,500 rpm). The collected solids were washed again with ethyl alcohol several times before drying overnight in an oven. The results of TEM and XRD analysis are presented in FIG. 2 and FIG. 3, respectively.

(22) <1-2> Preparation of Cross-Shaped Manganese Oxide Nanoparticles

(23) Manganese (II) acetate (1.4 mmol), oleylamine (3.0 mmol), oleic acid (1.5 mmol) and trioctylamine (6.2 ml) were loaded into a 100 ml Schlenk tube. The Schlenk tube was heated in an oil bath to 270° C. with a heating rate of 18° C. per minute and kept at that temperature for 1 hour with magnetic stirring and argon gas flow. Then oleic acid (2.4 mmol) and trioctylamine (1.24 ml) were injected to the reaction mixture followed by further heating at the temperature of 270° C. for 1 h. The green solids were obtained by cooling the reaction solution down to room temperature and washed with 1-propanol followed by a centrifugation (3 min, 3,500 rpm). The collected solids were washed again with ethyl alcohol several times before drying overnight in an oven. The result of TEM analysis is presented in FIG. 4.

(24) <1-3> Preparation of Urchin-Shaped Manganese Oxide Nanoparticles

(25) 6.2 ml trioctylamine, 1.4 mmol manganese (II) acetate, 3 mmol oleylamine and 1.5 mmol oleic acid were added to 100 ml Schlenk tube. The Schlenk tube was heated to 270° C. at a rate of 18° C./min in an oil bath under nitrogen blanket (the N.sub.2 gas was blown at the flow rate of 40 cc/min). After 1 h at 270° C., the formation of large MnO nanoparticles was completed. Then the formed large polycrystalline MnO nanoparticles were subjected to facet-selective etching. Specifically, in order to affect the anisotropic etching, oleic acid (1.6 mmol) and trioctylamine (1.24 ml) was injected to the reaction mixture, and the resulting solution was further heated at 270° C. for 1 h. The reaction mixture was cooled to room temperature, and excess ethanol was added into the solution to give a brown precipitate. The result of TEM analysis is presented in FIG. 5.

(26) <1-4> Preparation of Cubic Manganese Oxide Nanoparticles

(27) Manganese (II) acetate (0.4 mmol), sodium oleate (0.4 mmol) oleylamine (3.0 mmol), oleic acid (1.5 mmol) and trioctylamine (6.2 ml) were loaded into a 100 ml Schlenk tube. The Schlenk tube was heated in an oil bath to 270° C. with a heating rate of 18° C. per minute and kept at that temperature for 3 hour with magnetic stirring and argon gas flow. Then oleic acid (2.4 mmol) and trioctylamine (1.24 ml) were injected to the reaction mixture followed by further heating at the temperature of 270° C. for 1 h. The green solids were obtained by cooling the reaction solution down to room temperature and were washed with 1-propanol followed by a centrifugation (3 min, 3,500 rpm). The collected solids were washed again with ethyl alcohol several times before drying overnight in an oven. The result of TEM analysis is presented in FIG. 6.

Example 2: Preparation of Iron Oxide Nanoparticles with a Central MnO Phase

(28) <2-1> Preparation of Iron Oxide Nanoparticles with a Central MnO Core Using Octahedral Manganese Oxide Nanoparticles

(29) 14.2 mg of the octahedral MnO nanoparticles and 0.375 mmol of iron (III) acetylacetonate were added into the solution of oleic acid (0.05 mmol), oleylamine (1 mmol) and trioctylamine (2 ml) in a 100 ml Schlenk tube. The Schlenk tube was heated in the oil bath to 210° C. with the heating rate of 10° C. per min under vigorous stirring and kept at this temperature for 20 min under argon. Then the reaction mixture was heated at 310° C. for 30 min under dry air environment (oxygen percentage is 20%). The black solution was cooled to room temperature. After cooling down to room temperature, the iron oxide nanoparticles with a central MnO phase core were precipitated with an addition of acetone and n-propanol and were collected by centrifugation (3 min, 3,500 rpm). The obtained nanoparticles were washed several times in hexane and ethanol. A diagram illustrating the synthesis scheme of the iron oxide nanoparticles with a central MnO core is presented in FIG. 7. The results of TEM and XRD analysis of the iron oxide nanoparticles with a central MnO core are presented in FIG. 8 and FIG. 9, respectively. The result of electron energy loss spectroscopy (EELS) analysis of the iron oxide nanoparticles with a central MnO core is presented in FIG. 10.

(30) The resultant nanoparticles could be re-dispersed in chloroform, hexane or toluene for further using.

(31) <2-2> Preparation of Iron Oxide Nanoparticles with a Central MnO Core Using Various Manganese Oxide Nanoparticles

(32) To examine the applicability of the present invention, the preparation of iron oxide nanoparticles with a central MnO core using various manganese oxide nanoparticles was performed. In these experiments, iron oxide nanoparticles with a central MnO core were prepared by the same manner as performed to prepare iron oxide nanoparticles with a central MnO core using octahedral manganese oxide nanoparticles in the above, except cross-shaped, urchin-shaped or cubic MnO nanoparticles were used instead of octahedral manganese oxide nanoparticles.

(33) The results of TEM analysis of the iron oxide nanoparticles with a central MnO core are presented in FIGS. 11-13.

(34) The resultant nanoparticles could be re-dispersed in chloroform, hexane or toluene for further using.

Example 3: Preparation of Pyrenyl Polyethylene Glycol (Pyrenyl PEG)

(35) Pyrenyl polyethylene glycol (pyrenyl PEG) was synthesized by conjugating the amino group of hetero-functional polyethylene glycol (NH.sub.2—PEGCOOH, MW: 5,000 Da) with the n-hydroxysuccinimide (NHS) group of 1-pyrenebutyric acid n-hydroxysuccinimide ester (Py-NHS, Mw: 385.41 Da). In detail, 3 mmol of Py-NHS and 1 mmol of NH.sub.2-PEG-COOH were dissolved in 15 ml of dimethyl formamide, and then 200 μl of triethylamine was added to the reaction mixture at room temperature. After reacting for 48 hours at room temperature under a nitrogen atmosphere, the resultant products were filtered and washed with excess ether. The precipitates were dried under a vacuum and stored for later use.

Example 4: Coating Iron Oxide Nanoparticles with Pyrenyl Polyethylene Glycol (Pyrenyl PEG)

(36) The solution of iron oxide nanoparticles with a central MnO core in 1 ml of tetrahydrofuran (THF) was injected into 50 ml of phosphate buffer (pH 9.8) containing 300 mg of pyrenyl PEG. The resulting suspension was stirred overnight at room temperature to evaporate the organic solvent and subsequently centrifuged for 45 min at 20,000 rpm three times. After the supernatant was removed, the precipitates of iron oxide nanoparticles coated with pyrenyl PEG were re-dispersed in 10 ml of phosphate buffered saline (PBS; pH 7.4).

Example 5: Preparation of an MRI Contrast Agent Conjugated with Antibody

(37) For efficient targeting, an MRI contrast agent prepared in Example 4 was conjugated with antibody. In detail, 10 μmol of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 10 μmol of sulfo-n-hydroxysuccinimide (sulfo-NHS) as cross-linkers were added in 5 ml of the MRI contrast agent solution prepared in Example 4. And then, 0.7 mg (4.5 nmol) of anti HER2/neu antibody (Herceptin®; HER, Roche Pharmaceutical Ltd.) was added. The mixture was allowed to stand at 4° C. After 6 h, the MRI contrast agents conjugated with antibody (HER-conjugated MRI contrast agent) were purified by centrifugation (20,000 rpm, 45 min). Similarly, the irrelevant human immunoglobulin G (IgG) antibody (IRR) was conjugated with the MRI contrast agents by the same manner as performed to prepare the HER-conjugated MRI contrast agents in the above, except IRR was used instead of HER. The prepared IRR-conjugated MRI contrast agent was used as control MRI contrast agents without targeting molecule.

Example 6: T2-Weighted MR Imaging

(38) 0.5 ml of HER-conjugated MRI contrast agents were administered to nude mice. And then MR imaging was performed using a 3T clinical MRI instrument with a micro-47 surface coil (Philips Medical Systems, The Netherlands). The T2-weighted MR images of nude mice injected with HER-conjugated MRI contrast agents at 3T were acquired using the following measurements at room temperature: TR=4,000 milliseconds even echo space, number of acquisitions=1, point resolution of 312×312 μm, section thickness of 0.6 mm and TE=60 msec. The results are shown in FIG. 14.

(39) The result in FIG. 14 confirmed that the MRI contrast agent of the present invention could be used as an effective T2 MRI contrast agent.

Example 7: T1-Weighted MR Imaging

(40) 0.5 ml of HER-conjugated MRI contrast agents were administered to nude mice. And then MR imaging was performed using a 3T clinical MRI instrument with an 11×14 cm SENSE flex M coil having two elliptical elements (Philips Medical Systems, The Netherlands). The T1-weighted MR images of nude mice injected with HER-conjugated MRI contrast agents at 3T were acquired with selected echo time of 0.07 ms. The results are shown in FIG. 15.

(41) The result in FIG. 15 confirmed that the MRI contrast agent of the present invention could be used as an effective T1 MRI contrast agent.

(42) Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.