Magnetic nanoparticles comprising Gadolinium and method of fabrication

Magnetic nanoparticles are applicable in imaging, diagnosis, therapy, and biomaterial separation. The magnetic nanoparticles are represented as (FewGdx)vZy, wherein w is from 99.9% to 97.5%, x is from 0.1% to 2.5%, Z is an element of the group VIa, and v, y are positive numbers.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to magnetic nanoparticles applicable in imaging, diagnosis, therapy and biomaterial separation, and more particularly to magnetic nanoparticles suitable for use as contrast agents in Magnetic Resonance Imaging and a fabrication method thereof.

2. Description of the Related Art

In the biotechnology field, magnetic nanoparticles are applicable in imaging, diagnosis, therapy, biomaterial separation and so on. It is used, for example, in imaging as a contrast agent or a tracer to enhance the imaging contrast or to trace the presence of a certain disease. Furthermore, magnetic nanoparticles are also applicable in drug delivery and cancer therapy.

Currently, a number of image analysis techniques such as Computer Topography (CT), Magnetic Resonance Imaging (MRI), and ultrasound (US) are applied in disease diagnosis. The popular analysis technique of computer topography employs an X-ray to image for example, a human body by X-ray diffraction of various tissues with various densities. In addition, a contrast agent may be added during analysis to enhance the contrast among different tissues or organs. However, the radiation of X-rays may bring undesired side effects, thus Magnetic Resonance Imaging (MRI) has been provided as an alternative analysis technique.

Magnetic resonance imaging is capable of showing selectively image several different characteristics of tissues. The level of tissue magnetization at specific signal recording times during the MR imaging cycle generally determines the brightness of a particular tissue in the MRI images. Contrast is produced when tissues do not have the same level of magnetization. There are three primary magnetic characteristics of tissue that are the source of image contrast. Two of these are associated with the longitudinal magnetization. They are proton density and T1, the longitudinal relaxation time. The third characteristic is associated with the transverse magnetization. It is T2, the transverse relaxation time.

Diagnosis of brain disorders has been markedly improved by using MRI, which can delineate detailed anatomic structures with excellent tissue contrast on T1, T2, and proton density-weighted images; however, the inherent tissue characteristics do not always produce adequate contrast for some clinical applications. The administer materials that will alter the magnetic characteristics within specific tissues or anatomical regions, and can disclose abnormal enhancement after intravenous administration of contrast agents due to brain-blood-barrier (BBB) disruption. Advanced MR imaging technique, which can detect in vivo physiological changes in human brain, such as water diffusion, blood volume and blood flow have been implemented in clinical MR scanners.

Certain materials are susceptible to magnetic field and become magnetized when located in field. The orbital electrons in the atom rather than magnetic properties of the nucleus determine the susceptibility of a material. Contrast agents used in MRI are generally based on susceptibility effects. Using dynamic susceptibility contrast technique takes the advantage of T2 signal changes during the first-pass of a bolus of contrast agents. Hemodynamic parameters can then be calculated in terms of cerebral blood volume (CBV), cerebral blood flow (CBF) and mean transit time (MTT) for diagnosis in clinical.

MRI provides a non-invasive diagnosis. An MRI with contrast agent enhancement increases sensitivity and specificity of imaging in many cases particularly when relaxation times among different tissues are similar.

MRI contrast agents can be classified differently according to their magnetic properties (paramagnetic, ferromagnetic or superparamagnetic). However, current commercial MRI contrast agents employing magnetic nanoparticles have poor specificity and their contrast enhancement could be improved.

U.S. Pat. No. 5,427,767 discloses iron oxide doped with isotope including155Gd,156Gd, or157Gd in. Pure isotope, however, costs much higher than a nature isotope mixture. Further, the doping ratio and its effect of improving magnetization or transverse relaxivity (r2) are not discussed.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide magnetic nanoparticles, applicable in imaging, diagnosis, therapy, biomaterial separation, thereby furthering development of its application as an MRI contrast agent.

Therefore, by utilizing magnetic nanoparticles with Gd or forming an outer shell of Gd or its compound around the magnetic nanoparticles, the invention provides magnetic nanoparticles. The magnetic nanoparticles can be selectively modified by at least one molecule (such as liposome, polymer, aliphatic compound or aromatic compound), or further react with at least one substance having specificity (such as an antibody, protein, peptide, enzyme, carbohydrate, glycoprotein, nucleotide or lipid) to form contrast agents or tracers with specificity. Furthermore, the magnetic nanoparticles having specificity can perform a specific therapy such as killing cancer cells without harming healthy cells after entering the patient by heat transferred from the external magnetic field.

According to the invention, the provided magnetic nanoparticles are represented as (FewGdx)vZy, wherein w is from 0.999 to 0.975, x is from 0.001 to 0.025, Z is an element of the group VIa, and v, y are positive numbers.

The invention also provides a method of fabricating Gd-including iron oxide nanoparticles, comprising: (a) charging Gd and Fe ion salts in deionized water to form a mixture; (b) adjusting the pH value of the mixture to form precipitates.

The invention further provides a magnetic nanoparticle represented as FexMavZy, wherein Z is an element of the group VIa, x is greater, or equal to 0, and v, y are positive numbers, Mais an inner-transition element other than Gd.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, the provided magnetic nanoparticles may further have a core-shell structure as shown inFIG. 1, in which the core1-A is represented as FexMavZywhile the shell1-B is made of an inner-transition element Mbor the compound thereof. Similarly, Mais an inner-transition element, Z is an element of the group VIa, x is greater than or equal to 0, while v and y are positive numbers. Maand Mbmay be the same or different elements.

According to the invention, the element Z is, for example, oxygen or sulfur.

The invention also provides magnetic nanoparticles presented as (FewGdx)vZy, wherein w is from 0.999 to 0.975, x is from 0.001 to 0.25, Z is an element of the group VIa, and v, y are positive numbers. In preferred embodiments, the magnetic nanoparticles may have a transverse relaxivity (r2) of about 300 to 600 (mM Fe□s)−1.

The invention also provides a method of fabricating Gd-including iron oxide nanoparticles, comprising: (a) charging Gd and Fe ion salts in deionized water to form a mixture; and (b) adjusting the pH value of the mixture to form precipitates. When performed in air, in step (a) mixing ratio of Gd ion salt/(Gd ion salt+Fe ion salt) is about 0.1 to 99 mol %, preferably about 0.1 to 3 mol %, and more preferably about 2.5 mol %. When steps (a) and (b) are performed under inert gas, the method further comprising a step (c) oxidizing the precipitates in an acidic solution to form Gd-including iron oxide nanoparticles; and in step (a) mixing ratio of Gd ion salt/(Gd ion salt+Fe ion salt) is about 0.1 to 99.9 mol %, preferably about 2.5-10 mol %, and more preferably about 5 mol %. The resulting Gd-including iron oxide nanoparticles preferably have a transverse relaxivity (r2) of about 300 to 600 (mM Fe·s)−1.

The invention further provides a magnetic nanoparticle represented as FexMavZy, wherein Z is an element of the group VIa, x is greater, or equal to 0, and v, y are positive numbers, Mais an inner-transition element other than Gd.

According to the invention, the magnetic nanoparticles can be further modified by at least one molecule, such as a liposome, polymer, aliphatic compound, aromatic compound or combinations thereof.

The modified magnetic nanoparticles may further react with at least one substance having specificity, such as an antibody, a protein, a peptide, an enzyme, a carbohydrate, a glycoprotein, a nucleotide or a lipid. In addition, the substances with specificity may directly react with the unmodified magnetic nanoparticles to give specificity thereto.

EMBODIMENT

In the embodiment, magnetic nanoparticles of iron oxide doped with Gadolinium were given as an example, and the compound of the Gadolinium can be an oxide, sulfide, selenide, telluride, or polonide of the Gadolinium.

Preparation of Gd-Including Iron Oxide Nanoparticles in Air

In the embodiment, Gd-including iron oxide nanoparticles were prepared in air as an MRI contrast agent.

First, a reaction flask was charged with FeCl2powders (0.0069 moles), FeCl3powders (0.0138 moles) and deionized water (30 ml). FeCl3powders were replaced by GdCl3in various ratios in other examples. NaOH with a concentration of 5 M was added to control the pH value of the mixture. The mixture was subjected to continuous stirring during the reaction till the mixture became basic solution (the pH value approached about 11.5). Afterward, the temperature of the mixture was raised to and remained at 65° C. for 10 minutes. After black precipitates were formed, they were washed by deionized water and adjusted to acidic state by glacial acetic acid. Finally, H2O2(10 vol %) was gradually added until the end of the gaseous reaction, and was followed by a deionized water wash.

Characterization of Gd-Including Iron Oxide Nanoparticles

The magnetic nanoparticles prepared in air were then observed by TEM (JOEL, 100CX II).FIGS. 2a-2drespectively show the magnetic nanoparticles with an initial Gd3+/(Gd3++Fe2++Fe3+) mixing ratio of 0, 2.46, 3.33 and 6.67 mol %. In these cases, their average diameters are about 8.2±1.6 nm, 14.6±2.7 nm, 19.6±3.2 nm and 22.1±3.5 nm, respectively. The diameter of the nanoparticles is in direct proportion to initial Gd mixing ratio.

FIG. 3shows the XRD analysis of the magnetic nanoparticles prepared in air, further proving that the magnetic nanoparticles are iron oxide nanoparticles.

FIG. 4shows the ICP-AES analysis of the magnetic nanoparticles prepared in air. The magnetic nanoparticles with an initial Gd3+/(Gd3++Fe2+±Fe3+) mixing ratio of 0 mol %, 3.33 mol % or 6.67 mol % have a final Gd3+/(Gd3++Fe2++Fe3+) ratio in the nanoparticles of 0 mol %, 2.65 mol % or 3.20 mol %. The final Gd ratio is in direct proportion to the initial Gd mixing ratio.

FIG. 5shows the SQUID analysis of the magnetic nanoparticles prepared in air. The results indicate a 3-8% increased magnetization of the magnetic nanoparticles having 2.46 mol % of initial Gd mixing ratio.

After clinically injecting a contrast agent, the concentration of the contrast agent is diluted by blood or body fluid, so the effective concentration is less than the concentration of the commercial contrast agent. Therefore, the provided magnetic nanoparticles were prepared as a contrast agent having a concentration 2.5×10−3times that of a commercial MRI iron oxide contrast agent.FIG. 6shows the MRI analysis using the magnetic nanoparticles as a contrast agent. The longitudinal coordinates represent the signal intensity ratios of the oxides and water molecules. The greater the coordinates deviates from 1, the better the contrast enhancement is. As shown inFIG. 5, all of the four kinds of magnetic nanoparticles with various Gd initial mixing ratios exhibited contrast-enhancing capability. Especially, the iron oxide nanoparticles having 2.46 mol % additive GdCl3increased the contrast 18% more than that having non additive GdCl3under T2-weignted conditions. Note that the contrast enhancement is not in direct proportion to the initial mixing ratio. To the contrary, the maximum enhancement is provided when the initial mixing ratio is about 0.1-3 mol % when prepared in air, particularly about 2.5 mol %.

Preparation of Gd-Including Iron Oxide Nanoparticles Under Inert Gas

In another embodiment, Gd-including iron oxide nanoparticles were prepared under inert gas as an MRI contrast agent.

Under argon atmosphere, a reaction flask was charged with FeCl2powders (0.0345 moles), FeCl3powders (0.069 moles) and deionized water (150 ml) FeCl3powders were replaced by GdCl3in various ratios. NaOH with a concentration of 5 M was added to control the pH value of the mixture. The mixture was subjected to continuous stirring during the reaction till the mixture became basic solution (the pH value approached about 11.5). Afterward, the temperature of the mixture was raised to and remained at 65° C. for 10 minutes. After black precipitates were formed, they were washed by deionized water and adjusted to acidic state by glacial acetic acid. Finally, H2O2(10 vol %) was gradually added until the end of the gaseous reaction, and was followed by a deionized water wash. Note that before the precipitates were formed, all procedures were performed under argon.

The nanoparticles were dispersed in deionized water, dextran (Mw=10,000) is then added. After supersonic vibration, NH4OH was added to control the pH to 10. Continuously stirred, heated to 75□ and stirred at 75□ for 75 minutes. The suspension was dialyzed (using a membrane with molecular weight cutt off (MWCO) at 10,000) for removing excess dextran. The described suspension was put in a centrifuge at 6000 rpm for 30 minutes to remove the aggregates. Finally, the suspension is filtered over a filter of 0.2 μm in pore size to get surface modified nanoparticles.

Characterization of Gd-Including Iron Oxide Nanoparticles

The magnetic nanoparticles were then observed by TEM (JOEL, 2010).FIGS. 7a-7irespectively show the magnetic nanoparticles with an initial Gd3+/(Gd3++Fe2++Fe3+) mixing ratio of 0, 1, 2.5, 3, 5, 10, 20, 50, and 60 mol %. In these cases, their average diameters are about 12.4±3.9 nm, 12.7±3.0 nm, 13.2±3.1 nm, 16.7±3.8 nm, 18.9±4.3 nm, 34.0±8.3 nm, 48.3±11.0 nm, 76.1±16.1 nm, and 85.9±22.2 nm, respectively. The diameter of the nanoparticles is in direct proportion to initial Gd mixing ratio, as shown inFIG. 8.

FIG. 9shows the XRD analysis of the magnetic nanoparticles in the embodiment, further proving that the magnetic nanoparticles are iron oxide nanoparticles.

FIG. 10shows the ICP-AES analysis of the magnetic nanoparticles in the embodiment. The magnetic nanoparticles with an initial Gd3+/(Gd3++Fe2++Fe3+) mixing ratio of 0, 1, 2.5, 3, 5, 10, 20, 50, and 60 have a final Gd3+/(Gd3++Fe2++Fe3+) ratio in the nanoparticles of 0, 0.4±0.1, 0.8, 0.7, 1.0, 1.3±0.1, 1.4±0.1, 1.4±0.1, 1.5, and 1.4 mol %, respectively. Note that the final Gd ratio increases with the initial Gd mixing ratio before 20 mol %, but remains a constant after 20 mol %.

FIG. 11shows the SQUID analysis of the magnetic nanoparticles in the embodiment. The results indicate a 13% increased magnetization of the magnetic nanoparticles having 20 mol % of GdCl3added.

As shown inFIG. 12a, the magnetic nanoparticles which surface is modified by dextran with an initial Gd3+/(Gd3++Fe2++Fe3+) mixing ratio of 0, 1, 2.5, 3, and 5 mol % have a transverse relaxivity (r2) of 368±9, 424±1, 416±10, 370±3, and 480±12 (mM Fe·s)−1, respectively. As shown inFIG. 12b, the magnetic nanoparticles with an initial Gd3+/(Gd3++Fe2++Fe3+) mixing ratio of 0, 1, 2.5, 3, and 5 mol % have a longitudinal relaxivity (r1) of 39.2±0.7, 42.4±0.4, 39.0±1.2, 44.5±0.4, and 44.2±1 (mM Fe·s)−1. Note that commercial Resovist® has a transverse relaxivity of 204±1 (mM Fe·s)−1, and a longitudinal relaxivity of 26.3±0.3 (mM Fe·s)−1. Accordingly, the iron oxide nanoparticles having initial mixing ratio of 5 mol % additive GdCl3increased the r2 about 30% more than that having non additive GdCl3. Compared to commercial Resovist®, the r2 is significantly increased by about 2.35 times.

Compared to U.S. Pat. No. 5,427,767, a cheaper natural isotope mixture is used instead of pure isotope. Furthermore, the invention provides a detailed discussion of doping procedure and the effects of doping ratio to contrast enhancement.

Accordingly, the Gd-including iron oxide nanoparticles enhance the contrast effectively and provide a clearer MRI image. Furthermore, the provided Gd-including iron oxide nanoparticles may be selectively modified by a molecule such as a liposome, polymer, aliphatic compound, or aromatic compound. The modified magnetic nanoparticles may further react with a substance having specificity, such as an antibody, a protein, a peptide, an enzyme, a carbohydrate, a glycoprotein, a nucleotide or a lipid to form a contrast agent having specificity.