Patent Publication Number: US-2007122620-A1

Title: Nanoparticle-based imaging agents for x-ray / computed tomography and methods for making same

Description:
RELATED APPLICATIONS  
      This application is a continuation-in-part of U.S. patent application Ser. No. 11/265,728, filed Nov. 2, 2005, entitled “Nanoparticle-based Imaging Agents for X-Ray/Computer Tomography,” which is hereby incorporated herein by reference. 
    
    
     TECHNICAL FIELD  
      The present invention relates generally to imaging agents for use in X-ray/computed tomography, and more specifically to nanoparticle-based imaging agents and methods for making same.  
     BACKGROUND INFORMATION  
      Iodinated benzoic acid derivatives continue to serve as standard X-ray/computed tomography (CT) imaging agents, despite the risk factors and side effects associated with intravenous iodine injection. Additionally, such standard CT imaging agents are typically of low molecular weight, and they are known to clear from the human body very rapidly, making it difficult to target these agents to disease sites (Shi-Bao Yu and Alan D. Watson, Chem. Rev. 1999, 99, 2353-2377).  
      The literature describes experimental nanoparticle systems containing gadolinium (Gd) or iodine (I) for CT imaging. However, in such systems, only a relatively small number of heavy atoms may be delivered to/in the vicinity of the target tissues. Such approaches include a liposomal approach, in which iodinated molecules are encapsulated into liposomes (Leike et al., Invest. Radiol. 2001, 36(6), 303-308), as well as a dendritic approach, in which iodine atoms are conjugated to G-4 Starburst® polyamidoamide (PAMAM) dendrimers (Yordanov et al., Nano Letters 2002, 2(6), 595-599). Both approaches deliver, at most, a couple hundred heavy metal (i.e., gadolinium) atoms.  
      Efforts to deliver a greater number of heavy metal atoms have included the use of nanoparticles of such heavy metals. See PCT International Publication Nos. WO 03/075961 A2 and WO 2005/051435 A2. Although nanoparticles of elemental (zerovalent) metal species have the highest density (number of heavy metal atoms/volume), they suffer from issues such as robust synthesis and instability due to oxidation. Nanoparticles of inert metals such as gold (e.g., such as described in WO 03/075961 A2) can overcome these issues, but are not very cost effective.  
      As a result of the foregoing, there is a continuing need for new imaging agents for CT, especially to the extent that such imaging agents can provide for improved performance and benefit in one or more of the following areas: robust synthesis, reduced cost, image contrast enhancement, increased blood half-life, decreased toxicity, decreased radiation dose, and targeting capability.  
     BRIEF DESCRIPTION OF THE INVENTION  
      The present invention is generally directed to core/shell nanoparticles, wherein such core/shell nanoparticles comprise a nanoparticle core and a nanoshell disposed about the nanoparticle core such that, in the aggregate, they form a core/shell nanoparticle that is operable for use as an imaging agent in X-ray/computed tomography.  
      In some embodiments, the present invention is directed to an imaging agent comprising an active nanoparticle core comprising at least one heavy metal element in a non-zero valent state, and a passive nanoshell, the nanoshell being disposed about the nanoparticle core such that in the aggregate they form a core/shell nanoparticle that is operable for use as an imaging agent in CT imaging.  
      In some embodiments, an X-ray/computed tomography imaging solution comprises an ensemble of the imaging agents according to any of the embodiments described herein, wherein the mean diameter of the ensemble is not more than about 10 nm.  
      In some embodiments, the present invention is directed to methods of making any of the above-described imaging agents. In some or other embodiments, the present invention is directed to methods of using such imaging agents in CT.  
      In some embodiments, the present invention is directed to a method for making an X-ray/computed tomography imaging agent, the method comprising the steps of: providing a first precursor material comprising a heavy metal element; forming an active core from the first precursor material, the core comprising the heavy metal element in a non-zero valent state; providing a second precursor material; and forming a passive shell from the second precursor material, wherein the passive shell is disposed about the core such that the core and the shell form a core/shell nanoparticle.  
      The present invention uses a nanoparticle approach to deliver a relatively large number of high-density, highly-attenuating (radio-opaque molecular structures with effective atomic number greater than or equal to Z=34, the atomic number of selenium) atoms in elemental or molecular form to improve CT contrast enhancement. In some embodiments, the present invention provides for targeting of specific disease sites by the CT imaging agent. In some embodiments, the present invention provides for macrophage uptake of the CT imaging agent. In some embodiments, the present invention provides for a CT imaging agent with increased blood half-life.  
      The foregoing has outlined rather broadly the features of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention.  
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
      For a more complete understanding of the present invention, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:  
       FIG. 1  generally depicts a cross-sectional view of a core/shell nanoparticle so as to illustrate the relationship between the components;  
       FIG. 2  depicts a cross-sectional view of a core/shell nanoparticle comprising an active core and a passive shell, in accordance with some embodiments of the present invention;  
       FIG. 3  illustrates, in flow diagram form, a method of using core/shell nanoparticles as imaging agents for computed tomography, in accordance with some embodiments of the present invention;  
       FIG. 4  is a transmission electron microscope (TEM) image of active core/passive shell nanoparticles comprising a hafnium oxide core and polymeric shell, in accordance with some embodiments of the present invention;  
       FIG. 5  is a plot of the distribution of diameter of an ensemble of active core/passive shell nanoparticles comprising a tantalum oxide core and a polyethylene glycol (PEG) polymeric shell, in accordance some embodiments of the present invention;  
       FIG. 6  is a TEM image of active core/passive shell nanoparticles with tantalum oxide and a PEG polymeric shell, in accordance some embodiments of the present invention;  
       FIG. 7  is a plot of the distribution of diameter of an ensemble of active core/passive shell nanoparticles comprising a tantalum oxide core and a citrate ligand shell, in accordance some embodiments of the present invention;  
       FIG. 8  is a TEM image of active core/passive shell nanoparticles comprising a tantalum oxide core and a citrate ligand shell, in accordance some embodiments of the present invention;  
       FIG. 9  is a plot of the percentage of viable cells versus molar amount of tantalum for active core/passive shell nanoparticles with a tantalum oxide core and a PEG polymeric shell and for active core/passive shell nanoparticles with a tantalum oxide core and a citrate ligand shell, in accordance with some embodiments of the present invention;  
       FIG. 10  is a plot of a C3a standard curve to detect C3a in solution in accordance with some embodiments of the present invention;  
       FIG. 11  is a bar chart of C3a for active core/passive shell nanoparticles with a tantalum oxide core and each of the following shells: a glucose shell, an EDTA shell, and PEG shell in accordance with some embodiments of the present invention, and for various controls.  
       FIG. 12  is a computed tomography image of a rat injected with active core/passive shell nanoparticles with a tantalum oxide core and a PEG shell, in accordance with some embodiments of the present invention;  
       FIG. 13  is a series of computed tomography images at different times of a rat injected with active core/passive shell nanoparticles with a tantalum oxide core and a PEG shell, in accordance with some embodiments of the present invention. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      The present invention is generally directed to core/shell nanoparticles, wherein such core/shell nanoparticles comprise a nanoparticle core and a nanoshell disposed about the nanoparticle core such that, in the aggregate, they form a core/shell nanoparticle that is operable for use as an X-ray imaging agent, particularly in CT.  
      Referring to the drawings in general, it will be understood that the illustrations are for the purpose of describing a particular embodiment of the invention and are not intended to limit the invention thereto.  
      While most of the terms used herein will be recognizable to those of skill in the art, the following definitions are nevertheless put forth to aid in the understanding of the present invention. It should be understood, however, that when not explicitly defined, terms should be interpreted as adopting a meaning presently accepted by those of skill in the art.  
      “Computed Tomography,” abbreviated “CT,” as defined herein and also known as computed axial tomography or computer-assisted tomography (CAT) and body section roentgenography, is a medical imaging method employing tomography where digital processing is used to generate a three-dimensional image of the internals of an object (or subject) from a large series of two-dimensional X-ray images taken around a single axis of rotation. While the discussion herein focuses on computed tomography, it will be appreciated by those of skill in the art that such discussions apply generally to all types of X-ray imaging.  
      “Imaging agents,” as defined herein and also known as contrast agents, are agents that comprise a material that can significantly attenuate incident X-ray radiation causing a reduction of the radiation transmitted through the volume of interest. After undergoing CT image reconstruction and typical post-processing, this increased X-ray attenuation is interpreted as an increase in the density of the volume of interest, which creates a contrast enhancement in the volume comprising the contrast agent relative to the background tissue in the image. Because the discussion herein is generally applicable to all forms of X-ray imaging, the imaging agents of the present invention are generally referred to herein as “X-ray/computed tomography imaging agents.” This term is used interchangeably with “computed tomography (CT) agents.” 
      In reference to the core and shell components of a core/shell nanoparticle, the terms “active” and “passive” refer to the components&#39; ability to create a contrast enhancement in CT imaging. A conventional CT scanner scans uses a broad spectrum of X-ray energy between about 10 keV and about 140 keV. Those skilled in the art will recognize that the amount of X-ray attenuation passing through of a particular material per unit length is expressed as the linear attenuation coefficient. At an X-ray energy spectrum typical in CT imaging, the attenuation of materials is dominated by the photoelectric absorption effect and the Compton Scattering effect. Furthermore, the linear attenuation coefficient is well known to be a function of the energy of the incident X-ray, the density of the material (related to molar concentration), and the atomic number (Z) of the material. For molecular compounds or mixtures of different atoms the ‘effective atomic number,’ Z eff , can be calculated as a function of the atomic number of the constituent elements. The effective atomic number of a compound of known chemical formula is determined from the relationship:  
               Z   eff     =       [       ∑     k   =   1     P     ⁢       w     f   k       ⁢     Z   k   β         ]       1   β               (     Eq   .           ⁢   1     )             
 
 where Z k  is the atomic number of simple elements, P is the total quantity of simple elements, and w fk  is the weight fraction of simple elements with respect to the total molecular weight of the molecule (related to the molar concentration). The optimal choice of the incident X-ray energy for CT imaging is a function of the size of the object to be imaged and is not expected to vary much from the nominal values. It is also well known that the linear attenuation coefficient of the contrast agent material is linearly dependent on the density of the material, i.e., the linear attenuation coefficient can be increased if the material density is increased or if the molar concentration of the contrast material is increased. However, the practical aspects of injecting contrast agent material into patients, and the associated toxicity effects, limit the molar concentration that can be achieved. Therefore, it is reasonable to separate potential contrast agent materials according to their effective atomic number. Based on simulations of the CT contrast enhancement of typical materials for a typical CT energy spectrum with a molar concentration of approximately 50 mM, it is estimated that materials with effective atomic number greater than or equal to 34 will yield appropriate contrast enhancement of about 30 Hounsfield units (HU), or 3% higher contrast than water. Therefore, we have defined potential CT contrast materials as being either passive, having an effective atomic number of less than 34 (i.e., Z eff &lt;34), or active, having an effective atomic number greater than or equal to 34 (i.e., Z eff ≧34). See, e.g., Chapter 1 in Handbook of Medical Imaging, Volume 1, Physics and Psychophysics, Eds. J. Beutel, H. L. Kundel, R. L. Van Metter, SPIE Press, 2000). 
 
      A “nanoparticle,” as defined herein, is a particle with an average diameter of between about 1 nm and about 500 nm, and can be used to refer to nanoparticle cores and core/shell nanoparticle aggregates. Such nanoparticles can be spherical or irregularly shaped, and, particularly on the smaller end of this size range, are differentiated from molecular complexes. A “nanoshell,” as defined herein, is the shell region of the core/shell nanoparticle. Such a nanoshell is disposed about the nanoparticle core in such a way that it may or may not conform to the topography of the underlying nanoparticle core.  
      As mentioned above, and referring to  FIG. 1 , in some embodiments the present invention is directed toward an imaging agent comprising: (a) a nanoparticle core  101 ; and (b) a nanoshell  102 , the nanoshell being disposed about the nanoparticle core such that, in the aggregate, they form a core/shell nanoparticle  100  that is operable for use as an imaging agent in computed tomography (generally, X-ray) imaging. While  FIG. 1  depicts a cross-section of a perfectly spherical core/shell nanoparticle, such core/shell nanoparticles can also be irregularly-shaped.  
      In some embodiments, the above-described imaging agent further comprises at least one targeting agent. Such targeting agents are useful for targeting the imaging agents to specific diseased regions of a subject&#39;s body. Typically, the targeting agent is an antibody (e.g., IgG) or other peptide, but can also be a nucleic acid (e.g., DNA, RNA) or other suitable chemical species. Generally, the targeting agent is attached to one or both of the nanoparticle core and the nanoshell disposed about the nanoparticle core. Such attachment typically comprises a linkage such as, but not limited to, a peptide linkage, a disulfide linkage, an isothiourea linkage, an isourea linkage, a sulfonamide linkage, an amine linkage, a carbamate linkage, an amidine linkage, a phosphoramidate linkage, a thioether linkage, an arylamine linkage, an aryl thioether linkage, an ether linkage, a hydrazone linkage, a traizole linkage, an oxime linkage, and combinations thereof. See, e.g., Chapter 2 in Bioconjugate Techniques. G. T. Hermanson, Academic Press, 1996.  
      In some embodiments, the above-described CT imaging agents are optimized for macrophage uptake via control of the core/shell nanoparticle surface charge and/or the presentation of functional groups which induce macrophage uptake (e.g., polyvinyl sulfate).  
      For the core/shell nanoparticles described herein for use as imaging/contrast agents in CT, the nanoparticle core typically has an average diameter of from about 1 nm to about 100 nm, more typically from about 2 nm to about 80 nm, and most typically from about 2 nm to about 20 nm. The nanoshell disposed about the nanoparticle core typically has an average thickness of from about 0.5 nm to about 100 nm. Accordingly, the aggregate of the nanoparticle core and the nanoshell of the imaging agent typically has an average diameter of from about 2 nm to about 500 nm, more typically from about 2 nm to about 100 nm, and most typically from about 3 nm to about 20 nm.  
      It will be understood that it may be desirable for the aggregate of the nanoparticle core and the nanoshell to be excretable by a mammalian kidney, typically a human kidney. Accordingly, the aggregate of the nanoparticle core and the nanoshell of the imaging agent may have an average diameter of from about 1 to about 20 nm, more typically from about 2 to about 12 nm, and most typically from about 3 nm to about 8 nm.  
      In some embodiments, an X-ray/computed tomography imaging solution comprises an ensemble of the imaging agents of according to any of the embodiments described herein, wherein the mean diameter of the ensemble is not more than about 10 nm, more typically not more than about 7 nm, most typically not more than about 6 nm.  
      Active Core/Passive Shell Nanoparticles  
      Referring to  FIG. 2 , in some embodiments, the present invention is directed to an imaging agent  200  comprising an active nanoparticle core  201  and a passive nanoshell  202 , the nanoshell being disposed about the nanoparticle core such that in the aggregate they form a core/shell nanoparticle  200  that is operable for use as an imaging agent in CT imaging.  
      The material of which the above-described nanoparticle core  201  is comprised is generally limited only in that it comprises an active material for contrast enhancement in CT (Z eff ≧34), where the active material comprises at least one heavy metal element in a non-zero valent state. Generally, the nanoparticle core comprises a material distinguishable from that of the nanoshell. The nanoparticle core typically comprises material such as, but not limited to, metal oxides.  
      It will be understood, that such active materials may comprise elements of high atomic weight (e.g., heavy metals). That is, suitable metals include, but are not limited to, heavy metals. Suitable heavy metals include, but are not limited to, gadolinium (Gd), samarium (Sm), neodymium (Nd), tungsten (W), tantalum (Ta), bismuth (Bi), hafnium (Hf), barium (Ba), dysprosium (Dy), and combinations thereof. Thus, the nanoparticle core may comprise material such as, but not limited to, a compound of a heavy metal, where suitable compounds include, but are not limited to, oxides. Suitable oxides include, but are not limited to, tungsten oxide, tantalum oxide, hafnium oxide, bismuth oxide, and combinations thereof. By way of example and not limitation, the effective atomic number, Z eff , for Ta 2 O 5  is about 69.  
      The material of which the nanoshell  202  is comprised is not particularly limited, but generally does not comprise active CT contrast agent material (Z eff &lt;34) and must generally be capable of being disposed about the nanoparticle core. Suitable such materials include, but are not limited to, ligands, oligomers, polymers, clusters, carbohydrate species, functionalized silica, and combinations thereof. For example, suitable shell materials include, but are not limited to, polyethylene glycol, polyethylene imine, polymethacrylate, polyvinylsulfate, polyvinylpyrrolidinone, citrate, malate, glycolate, silanes, and combinations thereof.  
      According to some embodiments, the material of which the nanoshell is made is water soluble. Alternatively or in combination, according to some embodiments, the material of which the nanoshell is made is biocompatible.  
      Methods of Making  
      In some embodiments, the present invention is directed to method of making any or all of the above-described types of imaging agents in CT applications.  
      For example, when the imaging agent comprises an active nanoparticle core and a passive nanoshell, such methods may comprise the steps of: (a) providing a first precursor material comprising a heavy metal element; (b) forming an active core from the first precursor material, the active core comprising the heavy metal element in a non-zero valent state; (c) providing a second precursor material; and (d) forming a passive shell from the second precursor material, wherein the passive shell is disposed about the core such that the core and the shell form a core/shell nanoparticle. In some embodiments, the passive shell comprises a water soluble material derived from the second precursor material. Alternatively, or in combination, in some embodiments, the method further comprises controlling the average diameter of the nanoparticle.  
      It will be understood that the order and/or combination of steps may be varied. Thus, according to some embodiments, steps (a), (b), (c), and (d) occur as sequential steps so as to form the nanoparticle from the active core and the second precursor. By way of example and not limitation, in some embodiments, the first precursor comprises the heavy metal in a non-zero valent state; wherein the core comprises an oxide of the heavy metal in the same non-zero valent state; and wherein step (b) comprises hydrolysis of the first precursor. According to some embodiments, the first precursor is a salt, such as an alkoxide or halide, of the heavy metal, and the hydrolysis proceeds upon combining the first precursor, an acid, water, or water analog such as deuterium oxide, in alcoholic solvent. The water, or water analog, initiates the hydrolysis. The molar ratio of the acid to the heavy metal may be selected so as to control the size of the nanoparticle core. According to some embodiments, when the core comprises an oxide of a heavy metal, a silicon-containing material is attached to the core via Si—O linkages. According to some embodiments, the siloxane includes, but is not limited to polymerizable groups. The polymerization may proceed via acid induced condensation polymerization. Alternatively, according to some embodiments, a polymer may be physi-sorbed on the core. According to any of the above-described embodiments, the polymer may be water soluble and biocompatible. Suitable polymers include, but are not limited to polyethylene glycol (PEG), polyethylene imine (PEI), polymethacrylate, polyvinylsulfate, polyvinylpyrrolidinone and combinations thereof. It will be understood that we contemplate equivalent methods for forming nanoparticles comprising non-zero valent metallic cores comprising alternative heavy metal compounds described herein. By way of example and not limitation, the above description has been used to form niobium oxide nanoparticles starting from niobium ethoxide, Nb(OEt) 5 .  
      Alternatively, according to some embodiments, steps (a) and (c) occur together such that steps (b) and (d) occur together so as to form the nanoparticle directly from the first and second precursors. By way of example and not limitation, according to some embodiments, the first precursor comprises the heavy metal in a zero valent state; wherein the core comprises an oxide of the metal; and wherein and steps (b) and (d) together comprise adding the first and second precursors to a basic aqueous solution. According to some embodiments, the basic solution has a pH of at least about 9. Direct formation of the core and shell in the same step, such as by combining steps (a) and (b) is an unexpected discovery. Further, we have surprisingly discovered that exchange reactions which are well known for metal nanoparticles, such as those involving citrate shell formation on gold nanoparticles, may be used for making ligand based shells disposed about non-metallic cores, using, for example, the present direct preparation. When the shell comprises a ligand, the first precursor may comprise a ligand reagent. For example, when the ligand is a carboxylic acid anion, such as citrate, the ligand reagent is the associated carboxylic acid. Suitable alternative ligand reagents include, but are not limited to, sugars, alcohols, polyaldehydes, and combinations thereof. Glucose is an example that allows for the preparation of very small nanoparticles. Other suitable reagents include, but are not limited to, glycolic acid and malic acid. According to some embodiments, the diameter of the nanoparticles is controlled via selection of the identity and/or amount of ligand.  
      In combination with any of the above-described embodiments, in some embodiments, a method of making a X-ray/computed tomography preparation (e.g. containing a plurality of imaging agents) comprises forming an ensemble of core/shell nanoparticles, wherein the mean diameter of the ensemble is not more than about 10 nm, more typically not more than about 7 nm, most typically not more than about 6 nm. It will be understood that according to some embodiments, the mean diameter is selected so as to render the nanoparticles in the ensemble substantially clearable by a mammalian kidney, such as a human kidney, in particulate form.  
      Methods of Using  
      In some embodiments, the present invention is directed to methods of using any or all of the above-described types of imaging agents in CT applications. Referring to  FIG. 3 , such methods typically comprise the steps of: (Step  301 ) providing a quantity of core/shell nanoparticles comprising an active computed tomography contrast agent material, the core/shell nanoparticles each comprising a nanoparticle core and a nanoshell disposed about the nanoparticle core; (Step  302 ) administering the core/shell nanoparticles to a mammalian subject; and (Step  303 ) irradiating the subject with X-rays, in accordance with computed tomography, such that the core/shell nanoparticles serve as an imaging agent. In many such embodiments, such core/shell nanoparticles further comprise targeting agents such that, as imaging agents, they can be targeted to specific diseased areas of the subject&#39;s body. In some embodiments, the above-described core/shell nanoparticles, to the extent that they persist in the blood, are blood pool agents.  
      In some such above-described methods of using the above-described imaging agents, the core/shell nanoparticles provide for a CT signal of generally at least about 5 Hounsfield units to at most about 5000 Hounsfield units, and more particularly at least about 100 Hounsfield units to at most about 5000 Hounsfield units.  
      The following examples are included to demonstrate particular embodiments of the present invention. It should be appreciated by those of skill in the art that the methods disclosed in the examples that follow merely represent exemplary embodiments of the present invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments described and still obtain a like or similar result without departing from the spirit and scope of the present invention.  
     EXAMPLE 1  
      This Example serves to illustrate how a CT imaging agent can be prepared, in accordance with some embodiments of the present invention. In this particular Example, a passive shell is linked to an active core of hafnium oxide.  
      Hafnia nanocrystals (HfO 2 ) were prepared from a suspension of hafnium oxychloride in ethanol. An organosilane-based coating was applied as follows: dilute 3-glycidoxypropyl(trimethoxysilane), GPTS was diluted using butanol (volume ratio 1:0.5) and pre-hydrolyzed step by addition of 0.1M HCl keeping the molar ratio of GPTS:H 2 O at 1:0.5. The resulting solution was subjected to vigorous stirring overnight at room temperature, then loaded with HfO 2  nanocrystals. See Ribeiro et al., Appl. Phys. Left. 2000, 77 (22), 3502-3504.  FIG. 4  is a TEM image of active core/passive shell nanoparticles comprising a hafnium oxide core and polymeric shell, in accordance with some embodiments of the present invention.  
     EXAMPLE 2  
      This Example serves to illustrate how a CT imaging agent can be prepared, in accordance with some embodiments of the present invention. In this particular Example, a polymeric passive shell is linked to an active core of tantalum oxide. Further, in this particular Example, the shell formed about the core after the core is formed.  
      This example illustrates preparation of Ta 2 O 5  nanoparticles for X-ray imaging: 34 ml of n-propanol, 0.44 ml isobutyric acid and 0.5 ml deuterium oxide were combined under nitrogen in the order specified and stirred for 30 minutes at room temperature. Tantalum ethoxide (1.87 g) was added in a drop-wise manner, albeit rapidly, and stirring continued under nitrogen for 18 hours. The tantalum ethoxide contains Ta(V), that is tantalum in the +5 valence state, a non-zero valence state. 2-[Methoxy(poly-ethylenoxy)propyl]trimethoxysilane (PEGsilane550, 4.832 g) was added to the stirred mixture as a 40 ml solution in n-propanol and the reaction was refluxed for 1 hour in air. Once cooled to room temperature, HCl was added (125 μl, 0.1 M) and the reaction was allowed to stir overnight. Deionized water (40 ml) was added, the mixture stirred, and then all volatiles were removed to obtain a clear, colorless-to-slightly yellow gel-like product. To purify the nanoparticles for intravenous injection, the product was solubilized in deionized water and filtered through a 100 nm membrane. Dialysis in water for 12 hours (3500-8000 MWCO dialysis tubing), followed by 100 nm filtration and subsequent removal of water via lyophilization, yielded an off-white precipitate approximately 22% wt/wt in tantalum. Solutions as high as 1.8 M in tantalum have been prepared using saline (0.9% NaCl).  
       FIG. 5  shows the distribution of diameters for an ensemble of the nanparticles made according to this Example, as measured by conventional DLS, demonstrating a mean diameter of the ensemble of 7 nm.  FIG. 6  shows a TEM image of nanoparticles made according to this Example.  
     EXAMPLE 3  
      This Example serves to illustrate how a CT imaging agent can be prepared, in accordance with some embodiments of the present invention. In this particular Example, a polymeric passive shell is linked to an active core of tantalum oxide. Further, in this particular Example, the shell formed about the core after the core is formed.  
      This example illustrates preparation of Ta 2 O 5  nanoparticles for X-ray imaging: 34 ml of n-propanol, 0.44 ml isobutyric acid, and 0.5 ml deuterium oxide were combined under nitrogen in the order specified and stirred for 30 minutes at room temperature. Tantalum ethoxide (1.87 g) was added in a drop-wise manner, albeit rapidly, and stirring continued under nitrogen for 18 hours. The tantalum ethoxide contains Ta(V), that is tantalum in the +5 valence state, a non-zero valence state. Next, diethylphosphatoethyltriethoxysilane (PHS, 3 g) was added to the mixture as a 40 ml solution in n-propanol and the reaction was refluxed for 1.5 hours in air. Once cooled to room temperature, ammonium hydroxide was added (250 μl 0.1 M) and the reaction was allowed to stir overnight. Deionized water (40 ml) was then added to the reaction with stirring, and then hydrochloric acid was added (10 ml, 1.2 M) and allowed to react for 2 days at 50° C. Upon cooling, the reaction was neutralized with ammonium hydroxide to pH ˜7-8 and filtered through a 100 nm membrane. All volatiles were removed to give the product. To purify the nanoparticles for intravenous injection, the product was solubilized in deionized water (pH 7.5-8), filtered through a 100 nm membrane, dialyzed in water for 12 hours (3500-8000 MWCO dialysis tubing), and lyophilized to yield a snow-white precipitate approximately 30% wt/wt in tantalum. IR (nujol mull, NaCl plates, cm −1 ): 1454 (very strong), 1413 (weak), 1376 (strong), 1296 (weak), 1274 (weak), 1218 (strong), 1170 (medium), 1029 (very strong), 964 (very strong), 782 (medium). NMR (D 2 O, ppm):  31 P, 37.4 (broad);  1 H (broadened resonances), 4.16, 1.88, 1.37, 0.85.  
     EXAMPLE 4  
      This Example serves to illustrate how a CT imaging agent can be prepared, in accordance with some embodiments of the present invention. In this particular Example, a citrate ligand passive shell is linked to an active core of tantalum oxide. Further, in this particular Example, the shell formed in conjunction with the core.  
      This example describes direct preparation of Ta 2 O 5  nanoparticles from tantalum powder (325 mesh, Aldrich). The tantalum powder contained Ta(0), that is, tantalum in a zero valence state. A solution was prepared by combination of 20 ml of 30% hydrogen peroxide and 5 ml of ammonium hydroxide solution (28-30% NH 3  in water). These were combined in a 2-neck flask under nitrogen and cooled with an ice-bath. Subsequently, the addition of Ta powder (5 mmol, 0.907 g) was made and the mixture was allowed to stir for 2 h (magnetic stirring may be used at small scale, but mechanical stirrer is more appropriate for larger scales). Then citric acid (15 mmol, 3.152 g) was added and a condenser was fitted to the flask. The slurry was heated to 80° C. for 18 h. The reaction was then allowed to cool and the remaining tantalum powder was filtered off through a medium grade glass frit. The filtrate was concentrated by removal of water through rotoevaporation. The remaining colored solution was then dialyzed with a 3.5K cutoff in an aqueous solution buffered to pH of 7.2. The resulting solution was analyzed for the nanoparticles. If desired, the nanoparticles may be isolated by lyophyllization.  
       FIG. 7  shows the distribution of diameters for an ensemble of the nanparticles made according to this Example as measured by conventional DLS, demonstrating a mean diameter of the ensemble of 7 nm.  FIG. 8  shows a TEM image of nanoparticles made according to this Example.  
     EXAMPLE 5  
      This Example serves to illustrate how a CT imaging agent can be used, in accordance with some embodiments of the present invention. In this particular Example, cell viability studies of active core/passive shell nanoparticles demonstrate no appreciable loss of cell viability over the studied time period.  
      THP1 monocytes (ATCC TIB-71) were diluted to final density of 1×10 6  cells/mL in RPMI 1640+10% FBS media and 500 uL of cells were aliquoted into each well of a 24 well plate. Sterile-filtered tantalum oxide nanoparticles (100 mM Ta) were pipetted into wells. For control wells, either 50 uL of sterile 0.9% saline (negative control) or 80 uL of 30% hydrogen peroxide (positive control) was added to cells. The plate was swirled gently after addition and cells were cultured under normal conditions (37° C., 5% carbon dioxide, 100% humidity).  
      Following incubation, cells were pipetted into centrifuge tubes and cells were centrifuged at 300×g for 5 minutes at 20 degrees C., and the media was gently aspirated off. The cells were washed with 500 uL of 1×PBS, gently vortexed and centrifuged at 300×g for 5 minutes at 20° C. The cells were washed and the buffer was gently aspirated off. Annexin V FLUOS assay buffer (containing incubation buffer, Annexin V-fluorescein and propidium iodide) was prepared according to manufacturer&#39;s protocol (Annexin V FLUOS kit, Roche) and added 100 uL to each tube. Tubes with cells were gently vortexed and incubated at room temperature for 15 minutes. An additional 300 uL of incubation buffer were added to each tube prior to flow cytometry analysis.  
      The following were measured: the number of cells that were positive for Annexin V-fluorescein alone (apoptotic cells), positive for Annexin V-fluorescein and propidium iodide (necrotic cells) or negative for both (viable cells).  
      Referring to  FIG. 9 , all tantalum oxide preparations up to 0.1 mM Ta show negligible number of apoptotic or necrotic cells indicating no loss of cell viability after 3-hour exposure to nanoparticles.  
     EXAMPLE 6  
      This Example serves to illustrate how a CT imaging agent can be used, in accordance with some embodiments of the present invention. In this particular Example, complement activation studies of active core/passive shell nanoparticles demonstrate no appreciable increase in C3a compared to untreated or saline-treated serums.  
      Human plasma and serum was purchased from a commercial vendor (Bioreclamation). To isolate serum, the whole blood was drawn into serum tubes and allowed to clot at room temperature for 30-45 minutes. The sample was centrifuged at 2000×g for 10 minutes and the serum was immediately frozen at −70° C. Samples were shipped overnight on dry ice and stored at −70° C.  
      Serum was thawed at 37° C. for a few minutes in a water bath. 250 uL of serum was added into individual wells of a 24-well plate. Sterile test samples (Ta agent, saline and cobra venom factor) were added to sera to a final volume of 300 uL. The 24-well plate was transferred to an incubator (37° C.) for 30 minutes with occasional agitation.  
      Referring to  FIG. 10 , following the manufacturer&#39;s protocols (C3a assay, Quidel), stock solutions and dilutions were prepared to generate a C3a standard curve and high/low C3a samples.  
      To stop C3a production after 30 minute incubation, 37.5 mM sodium citrate was added to each well and 0.5 uL of serum (with and without test samples) was transferred to 1.5 mL of kit sample buffer in Eppendorf tubes. The tubes were inverted 3 times gently to mix the serum and sample buffer, then 100 uL of the diluted serum was added to individual wells of the C3a test strips and incubated at room temperature for 60 minutes. The wells were washed three times with wash buffer (200 uL of wash buffer per wash, wash buffer in the well for 1 minute per wash). 100 uL of C3a conjugate solution was added and the resulting combination incubated for 60 minutes at room temperature. The wash protocol was repeated (three washes, 200 uL of wash buffer per wash, wash buffer in the well for 1 minute per wash) then 100 uL of substrate was added to each well. After incubating 15 minutes, 100 uL of stop solution was added and the absorbance was measured at 450 nm on a 96 well UV plate reader. C3a levels were calculated from C3a standard curve.  
       FIG. 11  shows data from analysis. In particular,  FIG. 11  shows run data from incubation of tantalum oxide cores with each of the following shells; PEG, EDTA, citrate, and glucose in serum. Though some compounds (Ta EDTA) lowered C3a levels, none showed an increase in C3a compared to either untreated serum or saline-treated serum. No significant increase in C3a levels is observed by addition of up to 100 mM Ta. Elemental analysis via ICP-MS was used to determine total Ta content.  
     EXAMPLE 7  
      This Example serves to further illustrate how a core/shell nanoparticle-based CT imaging agent can be used to enhance contrast, in accordance with some embodiments of the present invention.  
      All imaging studies were performed on a EXplore Locus microCT imaging system using the following parameters: 80 kV, 4 minute acquisition.  
      A female Dark Agouti rat approximately 160 g in body weight was anesthetized by IP injection of ketamine/diazepam. An IV catheter (24 gage) was placed in the tail vein and flushed with 200 uL of sterile saline. The rat was then imaged on microCT from base of rib cage to bladder (Pre-injection scan). Immediately after the scan, the rat was injected via catheter with 1 mL of 210 mg Ta/mL tantalum oxide-PEG550 agent and scanned following completion of previous scan.  
      Referring to  FIG. 13 , images were reconstructed at half resolution and viewed using maximum intensity projections. Immediately following injection, contrast was observed in abdominal aorta, vena cava and renal arteries. Within 14 minutes after injection, the agent was visible in kidney and ureters as it was excreted to bladder. Bladder contrast was prominent within 23 minutes after injection. Still referring to  FIG. 13 , CT imaging studies show blood clearance between 6-14 minutes, followed by renal excretion to the bladder within 30 minutes.  
      The rat is imaged for 60 minutes after injection then returned to cage. After 24 hours, the rat is imaged again and no remaining contrast was observed in bladder or kidneys. No adverse effects are observed in injected rats.  
       FIG. 12  shows an abdominal artery image that was obtained by microCT imaging using nanoparticles with Ta 2 O 5  and PEG shells.  
      It will be understood that certain of the above-described structures, functions, and operations of the above-described embodiments are not necessary to practice the present invention and are included in the description simply for completeness of an exemplary embodiment or embodiments. In addition, it will be understood that specific structures, functions, and operations set forth in the above-described referenced patents and publications can be practiced in conjunction with the present invention, but they are not essential to its practice. In addition, it will be understood, that unless otherwise described, the steps of a method may be performed in any combination and/or order, including, but not limited to simultaneously. It is therefore to be understood that the invention may be practiced otherwise than as specifically described without actually departing from the spirit and scope of the present invention as defined by the appended claims.