# Patent

## Title
Non-radioactive traceable metal isotope-enriched nanoparticles and method of their use for determining biodistribution

## Abstract

Composition of non-radioactive traceable metal isotope-enriched nanoparticles, and methods of their use for determining in-vivo biodistribution are provided. The methods comprise the steps of: (a) introducing the nanoparticles into the biological material, wherein the nanoparticles comprise at least one inorganic core, and the inorganic core comprises at least two metal isotopes in a predetermined ratio; wherein at least one metal isotope is enriched non-radioactive traceable metal isotope and (b) determining the distribution of the nanoparticles in the biological material based on the predetermined ratio of the metal isotopes.

## Background
BACKGROUND
The invention relates generally to a composition of non-radioactive traceable metal isotope-enriched nanoparticles, and methods of their use for determining in-vivo biodistribution. One or more of the embodiments relate to non-radioactive traceable metal isotope-enriched superparamagnetic iron oxide (SPIO) nanoparticle composition and methods of their use for determining in-vivo biodistribution.
Superparamagnetic iron oxide (SPIO) nanoparticles of 10 to 1000 nm diameter are used as contrast agents to increase proton relaxation times in tissue following injection into patients to provide contrast enhancement in magnetic resonance imaging (MRI). It is often necessary to determine the nature of distribution of the contrast agent within the body. These determinations require detailed pharmacokinetic knowledge of the biodistribution and clearance of the contrast agent in animals, as well as blood and plasma clearance in human.
Although the degree of contrast observed by MRI depends upon the amount of contrast agent in a given tissue, it is generally not possible to quantitiatively determine the absolute concentration of conventional SPIO agents in a tissue. This is in part because of the effects of the SPIO agent on proton relaxation times. The SPIO agent's relaxivity properties may vary by interactions with proteins, cells or other components of the body and by possible metabolism or degradation of the SPIO agent within the body. Additionally, at lower concentrations of the SPIO agents, imaging sensitivity associated with MRI is a further barrier to quantitatively determine the concentration of the SPIO agents in tissue.
Other methods for determining SPIO concentrations in-vivo include iron-sensitive dyes, fluorescent/radioactive reporter labels, and quantitative elemental analysis. Qualitative methods, to determine SPIO distribution in tissue, rely on treating tissue specimens with iron staining agents, and cannot quantitatively determine biodistribution of the SPIO-based injected agents. Moreover, iron staining dyes do not necessarily distinguish between endogenous iron and SPIO nanoparticle associated iron. Dye, fluorophore, radioactive or other labels conjugated to the agent, change the composition of the SPIO particle, and may therefore alter its in-vivo performance. Furthermore, the label may not remain associated with the SPIO in-vivo, and thus tracking the label may not accurately reflect the distribution of the SPIO particle itself. In human studies, the potential risk of exposure to radioactive materials is also another concern. Elemental analysis of tissue samples to determine iron concentration could be achieved, however, with conventional SPIO agents, there is no way to distinguish between endogenous iron and SPIO-associated iron in the same tissue samples.
Therefore, there is a need for a label-free, non-radioactive method to quantitatively measure the in-vivo biodistribution of the injected nanoparticles such as SPIO nanoparticles.
BRIEF DESCRIPTION
The composition and methods of the invention are directed to the problem associated with radioactive nanoparticles at least in part by using non-radioactive traceable metal isotope-enriched nanoparticles. A “cold” or stable isotope with a low natural abundance is used herein, to facilitate tracking of the nanoparticle in-vivo. Moreover, one or more of the embodiments of the method are adapted to quantitate the nanoparticle biodistribution. For example, one embodiment of the method determines non-radioactive traceable metal isotope-enriched superparamagnetic iron oxide (SPIO) nanoparticle concentration in biological samples using elemental analysis. In one embodiment, Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) may be used to determine the amount of non-radioactive traceable metal isotope-enriched nanoparticle (such as Iron (Fe) nanoparticle) distributed in organs. Isotopes such as 57Fe may be used as a suitable non-radioactive traceable metal isotope for biodistribution studies of the nanoparticles. However, it may often be required to enrich the nanoparticles with the suitable isotopes, especially when isotopes of lower natural abundance are used. In some embodiments, nanoparticles enriched with 57Fe may be provided to assay the biodistribution effectively.
One or more embodiments of the present invention comprise a non-radioactive traceable metal isotope-enriched nanoparticle composition. The nanoparticle comprises at least one inorganic core and at least one coating substantially covering the inorganic core. The inorganic core comprises at least one non-radioactive traceable metal isotope. The nanoparticle may have a diameter in a range of from about 1 nm to about 1000 nm. The coating may comprise a biocompatible material. The non-radioactive traceable metal isotope enrichment in the nanoparticle may be in a range of from about 1% to about 99%.
Another aspect of the present invention comprises a non-radioactive traceable metal isotope-enriched superparamagnetic nanoparticle. The superparamagnetic nanoparticle comprises at least one inorganic core and at least one coating substantially covering the inorganic core. The inorganic core comprises at least one non-radioactive traceable metal isotope. The inorganic core may comprise a superparamagnetic material. The superparamagnetic material may comprise a superparamagnetic iron oxide (SPIO). The non-radioactive traceable metal isotope may comprise 54Fe, 57Fe, 58Fe, or combinations thereof.
One or more of the embodiments describe methods of forming a plurality of non-radioactive traceable metal isotope-enriched nanoparticles. The method comprises the steps of: (a) combining a non-polar aprotic organic solvent, an oxidant, and a surfactant, to form a first solution; (b) adding at least one non-radioactive traceable metal isotope-enriched organometallic compound to the first solution, to form a second solution; and (c) heating the second solution to a temperature in a range of from about 30° C. to about 400° C., to form the plurality of nanoparticles. In one embodiment, each of the plurality of nanoparticles may be coated with a surfactant.
One or more embodiments provide one or more methods for determining the extent to which nanoparticles have been distributed within a biological material. One or more of these methods comprise the steps of: (a) introducing the nanoparticles into the biological material, and (b) determining the distribution of the nanoparticles in the biological material. The nanoparticles used herein comprise at least one inorganic core, wherein the inorganic core comprises at least two metal isotopes in a predetermined ratio, wherein at least one metal isotope is enriched non-radioactive traceable metal isotope. The distribution of the nanoparticles in the biological material may be determined by assaying the predetermined ratio of the metal isotopes present in the nanoparticles. The method may include elemental analysis to determine the distribution of the non-radioactive traceable metal isotopes.



DRAWINGS
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
FIG. 1 shows a schematic representation of an embodiment, of the non-radioactive traceable metal isotope-enriched nanoparticles of the invention.
FIG. 2 shows mass spectrum of regular (non-isotope enriched) Fe(CO)5.
FIG. 3 shows mass spectrum of 57Fe-enriched Fe(CO)5.



DETAILED DESCRIPTION
The following detailed description is exemplary and not intended to limit the invention of the application and uses of the invention. Furthermore, there is no intention to be limited by any theory presented in the preceding background of the invention or the following detailed description.
In the following specification and the claims which follow, reference will be made to a number of terms have the following meanings. The singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Approximating language, as used herein throughout the specification and claims, may be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term such as “about” is not to be limited to the precise value specified. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. Similarly, “free” may be used in combination with a term, and may include an insubstantial number, or trace amounts, while still being considered free of the modified term. For example, free of solvent or solvent-free, and like terms and phrases, may refer to an instance in which a significant portion, some, or all of the solvent has been removed from a solvated material.
One or more embodiments of the invention relate to composition of a nanoparticle. The nanoparticle comprises at least one inorganic core and at least one coating substantially covering the inorganic core. The inorganic core comprises at least one non-radioactive traceable metal isotope and the non-radioactive traceable metal isotope is enriched in the nanoparticle.
Nanoparticle as used herein refers to particles having an average particle size on the nano scale. A nanoparticle may have a largest dimension (for example, a diameter or length) in the range of from about 1 nm to about 1000 nm. In one embodiment, an average particle size of the nanoparticle may be in a range of less than about 1 nm. In one embodiment, an average particle size of the nanoparticle may be in a range of from about 1 nm to about 10 nm, from about 10 nm to about 25 nm, from about 25 nm to about 50 nm, from about 50 nm to about 75 nm, or from about 75 nm to about 100 nm.
As used herein, a nanoparticle may refer to a single particle or may include a plurality of particles. The plurality of particles may be characterized by one or more of average particle size, particle size distribution, average particle surface area, particle shape, or particle cross-sectional geometry.
A plurality of particles may have a distribution of particle sizes that may be characterized by both a number-average size and a weight-average particle size. The number-average particle size may be represented by SN=Σ(sini)/Σni, where ni is the number of particles having a particle size si. The weight average particle size may be represented by SW=Σ(sini2)/Σ(sini). When all particles have the same size, SN and SW may be equal. In one embodiment, there may be a distribution of sizes, and SN may be different from SW. The ratio of the weight average to the number average may be defined as the polydispersity index (SPDI). In one embodiment, SPDI may be equal to about 1. In one embodiment, SPDI may be in a range of from about 1 to about 1.2, from about 1.2 to about 1.4, from about 1.4 to about 1.6, or from about 1.6 to about 2.0. In one embodiment, SPDI may be in a range that is greater than about 2.0.
In one embodiment, the metal nanoparticle may include a plurality of particles having a particle size distribution selected from a group consisting of normal distribution, unimodal distribution, and bimodal distribution. Certain particle size distributions may be useful to provide certain benefits, and other ranges of particle size distributions may be useful to provide other benefits. A normal distribution may refer to a distribution of particle sizes with SPDI equal to 1. A unimodal distribution may refer to a distribution of particle sizes having the same particle size. In another embodiment, particles having two distinct size ranges (a bimodal distribution) may be included in the composition: the first range from about 1 nm to about 10 nm and the second range from about 20 nm to about 50 nm.
A nanoparticle may have a variety of shapes and cross-sectional geometries that may depend, in part, upon the process used to produce the particles. In one embodiment, a nanoparticle may have a shape that is a sphere, a rod, a tube, a flake, a fiber, a plate, a wire, a cube, or a whisker. A nanoparticle may include particles having two or more of the aforementioned shapes. In one embodiment, a cross-sectional geometry of the particle may be one or more of circular, ellipsoidal, triangular, rectangular, or polygonal. In one embodiment, a nanoparticle may consist essentially of non-spherical particles. For example, such particles may have the form of ellipsoids, which may have all three principal axes of differing lengths, or may be oblate or prelate ellipsoids of revolution. Non-spherical nanoparticles may alternatively be laminar in form, wherein laminar refers to particles in which the maximum dimension along one axis is substantially less than the maximum dimension along each of the other two axes. Such laminar nanoparticles may have a shape similar to the tabular silver halide. Non-spherical nanoparticles may also have the shape of frusta of pyramids or cones, or of elongated rods. In one embodiment, the nanoparticles may be irregular in shape. In one embodiment, the nanoparticle may consist essentially of spherical particles.
A nanoparticle may have a high surface-to-volume ratio. A nanoparticle may be crystalline or amorphous. In one embodiment, a single type (size, shape, and the like) of nanoparticle may be used, or mixtures of different types of nanoparticles may be used. If a mixture of nanoparticles is used they may be homogeneously or non-homogeneously distributed in the composition.
In one embodiment, the metal nanoparticle may be stable towards aggregate or agglomerate formation. An aggregate may include more than one nanoparticle in physical contact with one another, while agglomerates may include more than one aggregate in physical contact with one another. In some embodiments, the nanoparticles may not be strongly agglomerated and/or aggregated such that the particles may be relatively easily dispersed in the composition.
In one or more embodiments, the nanoparticles comprise at least one inorganic core, wherein the inorganic core comprises at least one natural metal isotope and at least one non-radioactive traceable metal isotope, and the non-radioactive traceable metal isotope is enriched in the nanoparticle.
As used herein, the term “natural metal isotope” refers to a stable metal isotope that is present in the nanoparticle in its naturally occurring state and is non-enriched. As an example, the different isotopes of iron have a natural abundance of 54Fe (5.84%), 56Fe (91.7%), 57Fe (2.11%) and 58Fe (0.28%). Accordingly, for a nanoparticle enriched with 57Fe, the natural metal isotope may include 54Fe, 56Fe, or 58Fe. In some embodiments, the abundance of the natural metal isotope may be used to determine the distribution of the nanoparticle in the biological material, and accordingly the natural metal isotope may be chosen such that its concentration may be determined by an elemental analysis technique.
In one embodiment, the inorganic core comprises at least one natural metal isotope of a metal selected from a group consisting of iron, manganese, copper, titanium, cadmium, cobalt, nickel, chromium, gadolinium, yttrium, zinc, molybdenum, vanadium, and derivatives thereof.
In one embodiment, the natural metal isotope may comprise of isotopes of transition metals, lanthanides, or combinations thereof. In one embodiment, the natural metal isotope comprises an isotope of at least one metal selected from a group consisting of iron, manganese, copper, titanium, cadmium, cobalt, nickel, chromium, gadolinium, yttrium, zinc, molybdenum, vanadium, and combinations thereof. In one embodiment, an inorganic core comprises a compound of a natural metal isotope. Suitable compounds may comprise of oxides, nitrides, carbides, sulfides, phosphides, selenides, tellurides, borides, or combinations thereof. In some embodiments, a natural metal isotope may include 54Fe or 56Fe.
As used herein, the term “non-radioactive traceable metal isotope” refers to a metal isotope that may be a cold or a stable isotope. A non-radioactive traceable metal isotope present in the nanoparticle is enriched. As used herein, the term “enriched non-radioactive traceable metal isotope” refers to a non-radioactive traceable metal isotope present in the nanoparticle at an abundance that is greater than its natural abundance. In one embodiment, the abundance of the non-radioactive traceable metal isotope in the nanoparticle may be characterized by its enrichment value. As used herein, the term “non-radioactive traceable metal isotope enrichment” refers to the relative percentage of the number of the non-radioactive traceable metal isotopes present in the nanoparticle compared to the total number of metal isotopes present in the nanoparticle. Thus, by way of example, a nanoparticle comprising an enriched 57Fe isotope comprises 57Fe in an abundance that is greater than 2.11%. Similarly, a nanoparticle comprising an enriched 54Fe isotope comprises 54Fe in an abundance that is greater than 5.84%.
In one embodiment, the non-radioactive traceable metal isotope enrichment may be in a range of from about 1% to about 99%. In another embodiment, the non-radioactive traceable metal isotope enrichment may be in a range of from about 2% to about 99%, from about 5% to about 80%, from about 10% to about 70%, or from about 20% to about 50%. In some embodiments, the nanoparticle may be enriched with at least about 10% non-radioactive traceable metal isotope. In other embodiments, the nanoparticle may be enriched with at least about 20% non-radioactive traceable metal isotope. In other embodiments, the nanoparticle may be enriched with at least about 50% non-radioactive traceable metal isotope. In other embodiments, the nanoparticle may be enriched with at least about 90% non-radioactive traceable metal isotope. In one embodiment, the non-radioactive traceable metal isotope enrichment in the nanoparticle may be in a range of from about 10% to about 20%.
In one embodiment, the non-radioactive traceable metal isotopes may comprise an isotope of transition metals, lanthanides, or combinations thereof. In one embodiment, the non-radioactive traceable metal isotope comprises an isotope of at least one metal selected from a group consisting of iron, manganese, copper, titanium, cadmium, cobalt, nickel, chromium, gadolinium, yttrium, zinc, molybdenum, and vanadium, and combinations thereof. In one embodiment, an inorganic core comprises a compound of a non-radioactive traceable metal isotope. Suitable compounds may comprise of oxides, nitrides, carbides, sulfides, phosphides, selenides, tellurides, borides, or combinations thereof. In some embodiments, the non-radioactive traceable metal isotope may include 54Fe, 57Fe, or 58Fe.
In some embodiments, the inorganic core comprises a single enriched non-radioactive traceable metal isotope. In another embodiment, the inorganic core comprises two or more enriched non-radioactive traceable metal isotopes. In embodiments, where the inorganic core comprises two or more enriched non-radioactive traceable metal isotopes, the enriched non-radioactive traceable metal isotopes may be of the same metal or of two or more different metals. For example, in one embodiment, the enriched non-radioactive traceable metal isotope may comprise of different isotopes of a single metal, for example isotopes of 54Fe, 57Fe, 58Fe or combinations thereof. In another embodiment, the enriched non-radioactive traceable metal isotope may comprise two or more different transition metals, for example different isotopes of Fe and Mn. In another embodiment, the enriched non-radioactive traceable metal isotope may be comprised of two or more different lanthanides, for example isotopes of Gd and Yb. In another embodiment, the enriched non-radioactive traceable metal isotope may be comprised of different isotopes of transition metals and lanthanides, for example isotopes of Fe and Gd.
In some embodiments, the non-radioactive traceable metal isotope may comprise a non-radioactive traceable iron isotope. In one embodiment, the non-radioactive traceable iron isotope comprises 54Fe, 57Fe, 58Fe or combinations thereof Depending on the iron isotope employed, the iron isotope may be suitably enriched in the nanoparticle. In some embodiments, the non-radioactive traceable iron isotope enrichment in the nanoparticle may be in a range of from about 1% to about 99%. In some embodiments, the nanoparticle may be enriched with at least about 10% non-radioactive traceable iron isotope. In other embodiments, the nanoparticle may be enriched with at least about 20% non-radioactive traceable iron isotopes. In other embodiments, the nanoparticle may be enriched with at least about 50% non-radioactive traceable iron isotopes. In some embodiments, the nanoparticle may be enriched with at least about 90% non-radioactive traceable iron isotopes. In one embodiment, the non-radioactive traceable iron isotope enrichment in the nanoparticle may be in a range of from about 10% to about 20%. In some embodiments, the non-radioactive traceable metal isotope may include 57Fe. In embodiments including 57Fe as non-radioactive traceable metal isotope, the enrichment of 57Fe in the nanoparticle may be in a range that is greater than 2.5%.
In one embodiment, the inorganic core may be covered with at least one coating substantially covering the core. In one embodiment, the coating may serve to stabilize the inorganic core, i.e., the coating may prevent one inorganic core from contacting an adjacent inorganic core, thereby preventing a plurality of such nanoparticles from aggregating or agglomerating as described herein. In one embodiment, the coating may be of a sufficient thickness to stabilize the inorganic core and prevent such contact. In one embodiment, the coating has a thickness in a range of from about 1 nm to about 50 nm. In another embodiment, the coating has a thickness in a range of from about 1.5 nm to about 3 nm.
As used herein, the term “substantially covering” means that a percentage surface coverage of the nanoparticle is in a range that is greater than about 20%. Percentage surface coverage refers to the ratio of nanoparticle surface covered by the coating to the surface area not covered by the coating. In some embodiments, the percentage surface coverage of the nanoparticle may be in a range of from about 20% to about 80%. In other embodiments, the percentage surface coverage of the nanoparticle may be in a range of from about 40% to about 60%. In another embodiment, the percentage surface coverage of the nanoparticle is in a range that is greater than about 90%.
The inorganic core may be covered with one or more coating. In embodiments, where the inorganic core may be covered with more than one coating, the coating may be of the same or of different material. In embodiments, where more than one coating is present, the biocompatible material may be used for at least one coating. In some embodiments, all the coatings may be biocompatible. The non-limiting examples of a biocompatible coating include organo-silane compounds, polyketal polymers, dextran, poly (L) lactic acid, polycaprolactone, polyurethane, or combinations thereof. In some embodiments, a biocompatible coating may include organo-silane compounds. In some embodiments, the organo-silane compounds may be further derivatized with polyethylene glycol (PEG), polyethylene imine (PEI), dextran, or combinations thereof.
In some embodiments, the coating may further comprise of a spacer. In one embodiment, the spacer may comprise of alkyl groups, aryl groups, substituted alkyl and aryl groups, heteroalkyl (e.g., oxo groups, alkyl amine group), or heteroaryl groups. In some embodiments, the spacer may comprise of p-aminobenzyl, substituted p-aminobenzyl, diphenyl, substituted diphenyl, alkyl furan (e.g, benzylfuran), carboxy, or straight chain alkyl groups of 1 to 10 carbons in length. In some particular embodiments, the spacer may comprise of p-amino benzyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, acetic acid, propionic acid, amino butyl, p-alkyl phenols, 4-alkylimidazole, carbonyls, OH, COOH, or glycols.
In some embodiments, the coating “substantially covering” the core may facilitate one or more of improved water solubility, reduces aggregate formation, reduces agglomerate formation, prevent oxidation of nanoparticles, maintain uniform core-shell entity, or biocompatibility of the nanoparticles. In another embodiment, the material or materials comprising the coating may later be replaced or functionalized by other materials that are tailored for a particular application, such as, but not limited to, diagnostic application.
In one embodiment, the nanoparticle may further be functionalized with a targeting moiety. The targeting moiety may be a molecule or a structure that provides targeting of the nanoparticle to desired organs, tissues or cells. The targeting moiety may include, but is not limited to, proteins, peptides, antibodies, nucleic acids, sugar derivatives, or combinations thereof.
In some embodiments, the nanoparticle may comprise a single inorganic core. In some embodiments, the nanoparticle may comprise a plurality of inorganic cores. In embodiments comprising plurality of inorganic cores, at least one inorganic core comprises an enriched non-radioactive traceable metal isotope. In some embodiments, at least two inorganic cores comprise an enriched non-radioactive traceable metal isotope. In other embodiments, each of the inorganic cores comprises an enriched non-radioactive traceable metal isotope. In some embodiments, at least one of the inorganic cores may not contain any non-radioactive traceable metal isotope.
In embodiments involving plurality of inorganic cores having an enriched non-radioactive traceable metal isotope, the non-radioactive traceable metal isotope may be of the same or of different metal in the different inorganic cores. In one exemplary embodiment, both a first and a second inorganic core may be enriched with 57Fe. In another exemplary embodiment, a first inorganic core may be enriched with 54Fe and a second inorganic core may be enriched with 57Fe. In another exemplary embodiment, a first inorganic core may be enriched with a non-radioactive traceable metal isotope of Fe and a second inorganic core may be enriched with a non-radioactive traceable metal isotope of Mn.
The inorganic cores may be covered with one or more coatings. In some embodiments, the plurality of inorganic cores may be covered with the same coating. In one embodiment, a single coating may cover all the inorganic cores present in the nanoparticle. In some embodiments, the individual cores may further be covered with one or more coatings. In another embodiment, all the inorganic cores present in the nanoparticle may be covered with two or more coatings. The individual coating may comprise the same material or may comprise two or more different materials.
In one or more embodiments, the present invention relates to a composition of a superparamagnetic nanoparticle. “The superparamagnetic nanoparticle” as used herein refers to nanoparticles that may exhibit a behavior similar to paramagnetism even when at temperatures below the Curie or the Néel temperature.
In some embodiments, the superparamagnetic nanoparticle composition comprises at least one inorganic core and at least one coating substantially covering the inorganic core. The inorganic core comprises at least one non-radioactive traceable metal isotope and the non-radioactive traceable metal isotope is enriched in the nanoparticle. In some embodiments, the inorganic core comprises a superparamagnetic material.
In some embodiments, the superparamagnetic nanoparticle comprises at least one natural metal isotope, and at least one non-radioactive traceable metal isotope in a predetermined ratio. In other embodiments, the superparamagnetic nanoparticle comprises at least one superparamagnetic iron oxide (SPIO) core. The SPIO core comprises a non-radioactive traceable metal isotope. In some embodiments, the non-radioactive traceable metal isotope in the SPIO core may comprise at least one isotope of iron, for example, 54Fe, 57Fe, or 58Fe. In other embodiments, the non-radioactive traceable metal isotope in the SPIO core may comprise at least one non-iron isotope, for example, a non-radioactive traceable isotope of Mn. In some embodiments, the SPIO core may include enriched non-radioactive traceable metal isotope. The non-radioactive traceable metal isotope enrichment may be in a range of from about 2% to about 99%. In some embodiments, the non-radioactive traceable metal isotope enrichment may be in a range of from about 60% to about 90%. In some other embodiments, the non-radioactive traceable metal isotope enrichment may be in a range of from about 10% to about 25%. In some embodiments, the non-radioactive traceable metal isotope may be 54Fe, 57Fe, or 58Fe.
In some embodiments, the superparamagnetic nanoparticle may have a diameter in a range of from about 1 nm to about 1000 nm. In one or more of the embodiments, the nanoparticle may have a diameter in a range of from about 1 nm to about 100 nm. In other embodiments, the nanoparticle may have a diameter in a range of from about 1 nm to about 10 nm.
In some embodiments, the superparamagnetic nanoparticle comprises a single SPIO core. The SPIO core may have one or more coating substantially covering the SPIO core. In embodiments, where more than one coating is present, the coating may be of the same material or of different materials. In some embodiments, the superparamagnetic nanoparticle comprises a single SPIO core and at least one additional core. In some embodiments, the superparamagnetic nanoparticle comprises one or more inorganic cores in addition to one or more SPIO cores.
In some embodiments, the superparamagnetic nanoparticle comprises a plurality of SPIO cores. The plurality of SPIO cores may have one or more coatings substantially covering the SPIO cores. In embodiments, where more than one coating is present, the coating may be of the same material or of different materials. In some embodiments, two or more of the SPIO cores may be covered with one and the same coating. In some embodiments, the individual cores may further be covered with one or more additional coatings. In some embodiments, some of the superparamagnetic iron oxide (SPIO) cores may not contain non-radioactive traceable metal isotopes. In some embodiments, each of the SPIO cores may comprise at least one non-radioactive traceable metal isotope.
In some embodiments, the nanoparticles of the present invention may be used as magnetic resonance (MR) contrast agents. These nanoparticles may yield a T2*, T2, or T1 magnetic resonance signal upon exposure to a magnetic field.
Another aspect of the invention is to provide a method for making a plurality of nanoparticles. The method comprises the steps of (a) combining a non-polar aprotic organic solvent, an oxidant, and a surfactant, to form a first solution, (b) adding at least one organometallic compound to the first solution, to form a second solution, and (c) heating the second solution to a temperature in a range from about 30° C. to about 400° C., to form the plurality of nanoparticles. The plurality of nanoparticles comprises at least one inorganic core derived from the organometallic compound. The inorganic core comprises at least one non-radioactive traceable metal isotope. The plurality of nanoparticles further comprises at least one coating derived from the surfactant.
In one embodiment, the non-polar aprotic organic solvent may be thermally stable at the temperature at which the plurality of nanoparticles is formed. In one embodiment, the non-polar aprotic solvent has a boiling point in a range of from about 275° C. to about 340° C. Suitable non-polar aprotic solvents include, but are not limited to, dioctyl ether, hexadecane, trioctylamine, tetrathylene glycol dimethyl ether (also known as “tetraglyme”), or combinations thereof.
In one embodiment, the oxidant comprises at least one organo-tertiary amine oxide, a peroxide, an alkyl hydroperoxide, a peroxy-acid, molecular oxygens, nitrous oxide, or combinations thereof In one embodiment, the oxidant comprises an organo-tertiary amine oxide having at least one methyl group. One non-limiting example of such an oxidant is trimethyl amine oxide.
In one embodiment, the surfactant comprises a polymerizable functionalized group, an initiating functionalized group, a cross-linking functionalized group, or combinations thereof. In one embodiment, the polymerizable functionalized group may comprise at least one of an alkene, an alkyne, a vinyl (including acrylics and styrenics), an epoxide, an azeridine, a cyclic ether, a cyclic ester, a cyclic amide, or combinations thereof. In one embodiment, the initiating functionalized group may comprise at least one of a thermal or a photoinitiator, such as, but not limited to, an azo compound, a hydroxide, a peroxide, an alkyl halide, an aryl halide, a halo ketone, a halo ester, a halo amide, a nitroxide, a thiocarbonyl, a thiol, an organo-cobalt compound, a ketone, an amine, or combinations thereof. In one embodiment, the cross-linking functionalized group may comprise at least one of a thiol, an aldehyde, a ketone, a hydroxide, an isocyanide, an alkyl halide, a carboxylate, a carboxylic acid, a phenol, an amine, or combinations thereof.
In one embodiment, the at least one organometallic compound comprises at least one non-radioactive traceable metal isotope and at least one ligand. In one embodiment, the organometallic compound comprises a non-radioactive traceable metal isotope of a transition metal, a lanthanide, or combinations thereof. In another embodiment, the organometallic compound comprises a non-radioactive traceable metal isotope of at least one metal selected from a group consisting of iron, manganese, copper, titanium, cadmium, cobalt, nickel, chromium, gadolinium, yttrium, zinc, molybdenum, vanadium, and combinations thereof.
A suitable ligand may comprise, but is not limited to, a carbonyl group, a cyclo octadienyl group, an organophosphine group, a nitrosyl group, a cyclo pentadienyl group, a pentamethyl cyclo pentadienyl group, a [π]-acid ligand, a nitroxy group, or combinations thereof. Non-limiting examples of the organometallic compound include iron carbonyl (Fe(CO)5), cobalt carbonyl (Co(CO)8), or manganese carbonyl (Mn2(CO)10). In one embodiment, an amount of the organometallic compound may be introduced into the aprotic solvent such that a ratio of the concentration of the organometallic compound to the concentration of the oxidant is from about 1:1 to about 10:1.
In one embodiment, the second solution may be heated to a first temperature for first time interval. The first temperature may be in a range of from about 50° C. to about 400° C. In one embodiment, the first temperature is in a range of from about 275° C. to about 400° C. and preferably, in a range of from about 275° C. to about 310° C. The length of the first time interval may be in a range of from about 30 minutes to about 2 hours, depending on the particular organometallic compounds and oxidants that are in the aprotic solvent. One skilled in the art, given the benefit of this disclosure will be able to derive appropriate temperature and time interval that are suitable for synthesizing the plurality of nanoparticles.
In one embodiment, the method may further comprise the step of: precipitating the plurality of nanoparticles from the non-polar aprotic solvent. Precipitation of the plurality of nanoparticles may be accomplished by adding at least one of an alcohol or a ketone to the non-polar aprotic solvent. Alcohols such as, but not limited to, methanol and ethanol may be used. Alcohols having at least three carbon atoms, such as isopropanol, are preferred. Ketones such as, but not limited to, acetone may be used in conjunction with, or separate from, an alcohol in the precipitation step.
In one embodiment, the method further comprises reacting or interacting the metal nanoparticles with a biocompatible material to form a biocompatible coating of the nanoparticle. In one embodiment, the biocompatible material either partially or completely replaces or is exchanged with the surfactant in the coating. Following the formation of the plurality of nanoparticles, the biocompatible material may be added to the nonpolar aprotic solvent such that the biocompatible material is present in a concentration, greater than the concentration of the surfactant in the nonpolar aprotic solvent. Non-limiting examples of biocompatible materials include organo-silane compounds, polyketal polymers, dextran, poly (L) lactic acid, polycaprolactone, polyurethane, or combinations thereof. In some embodiments, the biocompatible material may include organo-silane compounds. In some embodiments, the organo-silane compounds may be further derivatized with polyethylene glycol (PEG), polyethylene imine (PEI), dextran, or combinations thereof.
One embodiment of the invention describes the method for determination of the extent to which the nanoparticles are distributed within a biological material. The nanoparticles may be introduced to the biological material by a variety of known methods. Non-limiting examples for introducing the nanoparticle to the biological material include intravenous administration, oral, dermal application, or direct injection into muscle, skin, the peritoneal cavity or other tissues or bodily compartments. The method comprises the steps of: (a) introducing the nanoparticles into the biological material, wherein the nanoparticles comprise at least one inorganic core, and the inorganic core comprises at least two metal isotopes in a predetermined ratio, wherein at least one metal isotope is enriched non-radioactive traceable metal isotope, and (b) determining the distribution of the nanoparticles in the biological material based on the predetermined ratio of the metal isotopes.
As used herein, the term “predetermined ratio” refers to a ratio of the abundance of the two metal isotopes in the nanoparticle. In one embodiment, at least one metal isotope is a non-radioactive traceable metal isotope. In another embodiment, both the metal isotopes are non-radioactive traceable metal isotopes. As described herein above, the non-radioactive traceable metal isotope may be enriched in the nanoparticle. In another embodiment, one of the metal isotope is a natural metal isotope, which is non-enriched. Thus, in one embodiment, the predetermined ratio is the ratio of the abundance of the two enriched non-radioactive traceable metal isotopes. In another embodiment, the predetermined ratio is the ratio of the abundance of the enriched non-radioactive traceable metal isotope to the natural abundance of the natural metal isotope in the nanoparticle.
Another embodiment, of the invention describes the method for determination of the extent to which the superparamagnetic nanoparticles may be distributed within a biological material. The method comprises the steps of (a) introducing the superparamagnetic nanoparticles into the biological material, where the superparamagnetic nanoparticles comprise at least one superparamagnetic core, wherein the superparamagnetic core comprises at least two metal isotopes in a predetermined ratio; wherein at least one metal isotope is enriched non-radioactive traceable metal isotope, and (b) determining the distribution of the superparamagnetic nanoparticles in the biological material based on the predetermined ratio of the metal isotopes.
In another embodiment, the distribution of the nanoparticle comprising non-radioactive traceable metal isotopes in the biological material may be determined by elemental analysis. In one embodiment, Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) may be used to determine the concentration of the nanoparticles in the biological material.
In one embodiment, the predetermined ratio may be related to the distribution of nanoparticle in the biological material. In one embodiment, the distribution of nanoparticle in the biological material may be determined by measuring the ratio of two non-radioactive traceable metal isotopes present in the biological material. In another embodiment, the distribution of nanoparticle in the biological material may be determined by measuring the ratio of non-radioactive traceable metal isotope and natural metal isotope present in the biological material. The amount of natural metal isotope present in the biological material is the sum of the natural metal isotope present in the nanoparticle and the natural metal isotope already present in the biological material at its natural abundance. The amount of non-radioactive traceable metal isotope present in the biological material is sum of the enriched non-radioactive traceable metal isotope present in the nanoparticle and the non-radioactive traceable metal isotope already present in the biological material at its natural abundance.
In one exemplary embodiment, 57Fe-enriched SPIO nanoparticle (57FeSPIO) may be employed to determine 57FeSPIO concentration in biological samples for characterization of biodistribution and clearance of the nanoparticle. As described herein, 3 isotopic ratios of 57Fe to 54Fe may be employed to determine distribution of SPIO in the biological sample. One of the three ratios is the ratio of non-radioactive traceable iron isotopes (57FeSPIO to 54FeSPIO) in the superparamagnetic iron oxide (57FeSPIO) nanoparticle—(represented by ‘S’). The ratio of non-radioactive traceable iron isotopes (57FeSPIO to 54FeSPIO) may be measured by elemental analysis for each of the 57FeSPIO. Second of the three ratios is the ratio of natural abundance of the 57Fenatural to 54Fenatural in a tissue sample (represented by ‘N’). The third ratio is the ratio of the non-radioactive traceable iron isotopes (57Fesample to 54Fesample) (represented by ‘M’) measured in a biological sample by elemental analysis upon the biological sample being injected with 57FeSPIO.
As described hereinabove, M depends on both on the endogenous iron content of the tissue and on the concentration of 57Fe-enriched SPIO in the tissue. M may be represented by:




  
    
      
        M
        =
        
        ⁢
        
          
            Fe
            sample
            
            
              
              57
          
          
            Fe
            sample
            
            
              
              54
          
        
      
    
  
  
    
      
        =
        
        ⁢
        
          
            
              Fe
              SPIO
              
              
                
                57
            
            +
            
              Fe
              natural
              
              
                
                57
            
          
          
            
              Fe
              SPIO
              
              
                
                54
            
            +
            
              Fe
              natural
              
              
                
                54
            
          
        
      
    
  




The concentration of 57Fe in the sample attributable to 57FeSPIO may be then calculated as:




  
    Fe
    SPIO
    
    
      
      57
  
  =
  
    
      
        
          Fe
          sample
          
          
            
            57
        
        /
        M
      
      -
      
        
          Fe
          sample
          
          
            
            57
        
        /
        N
      
    
    
      
        1
        /
        S
      
      -
      
        1
        /
        N
      
    
  




Based on the 57Fe enrichment in the 57FeSPIO preparation (e.g., 57FeSPIO: SPIO (SPIO with no enrichment), the total iron concentration in the tissue attributable to 57FeSPIO may be calculated by:

SPIO distributed in the biological material=57FeSPIO/Enrichment

EXAMPLES
Practice of the invention will be still more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way.
The abbreviations used in the examples section are expanded as follows: “mg”: milligrams; “mL”: milliliters; “mg/mL”: milligrams per milliliter; “mmol”: millimoles; “μL” and μLs: microliters; “ICP-MS”: Inductively Coupled Plasma Mass Spectroscopy; “MALDI-MS”: Matrix Assisted Laser Desorption Ionization Mass Spectrometry; “HPLC”: High Pressure Liquid Chromatography; ppb: parts per billion; “TEM”: Transmission Electron Microscopy; “EDX”: Energy Dispersive X-ray Elemental Analysis, “XAS”: X-ray Absorption Spectroscopy; “SAED-XRD”: Selected Area Electron Diffraction-X-ray Diffraction Crystal Symmetry pattern Indexing.
Unless otherwise noted, all reagent-grade chemicals were used as received, and Millipore water was used in the preparation of all aqueous solutions. The 57Fe was purchased from Cambridge Isotope Laboratories, Inc. Andover Mass., as iron-57 oxide (57Fe>95% enrichment).
Example 1
Synthesis of 57Fe (CO)5




Iron-57 oxide (57Fe2O3) was converted to non-radioactive isotopically enriched iron-57 metal (57Fe) by heating the Iron-57 oxide at 700° C. under H2 atmosphere for 2 h. In a subsequent step, the iron-57 metal (57Fe) was subjected to carbonylation reaction at around 120° C. for at least 4 hours, under at least 100 atmospheric carbon monoxide (CO) pressure to obtain non-radioactive isotopically enriched iron-57 carbonyl (57Fe(CO)5). Typically, the 57Fe2O3 was combined with a larger portion of commercially available (regular) Fe2O3 for ease in materials handling to prepare 57Fe-enriched Fe(CO)5. The %-enrichment of Fe(CO)5 is then determined by the ratio of 57Fe2O3/Fe2O3 used.
FIG. 2 shows the mass spectrum of regular (non-isotope enriched) Fe(CO)5. The peak 56 corresponds to natural Fe isotope. Peak 57 corresponds to 57Fe present in the regular Fe(CO)5.
FIG. 3 shows the mass spectrum of 57Fe enriched Fe(CO)5 (57Fe(CO)5). The peak 56 corresponds to natural Fe isotope. Peak 57 corresponds to 57Fe present in the 57Fe(CO)5. The 57Fe peak intensity in FIG. 3 is higher than 57Fe peak intensity in FIG. 2. This clearly demonstrates the 57Fe enrichment in 57Fe(CO)5 compared to regular Fe(CO)5.
Example 2
Synthesis of Non-Radioactive 57Fe Enriched Superparamagnetic Iron Oxide (SPIO) Nanoparticle




Trimethyl-N-oxide, 10-undecanoic acid, lauric acid and dioctyl ether were each individually dehydrated and deoxygenated. A mixture of trimethylamine-N-oxide (7.60 mmol), 10-undecenoic acid (4.56 mmol) and 7 ml of dioctyl ether or a mixture of trimethylamine-N-oxide (7.60 mmol), lauric acid (4.56 mmol) and 7 ml dioctyl ether were added under an inert atmosphere to a 50 ml two neck Schlenk flask. The flask was attached to a Schlenk vacuum-line, and a column-reflux condenser assembly was attached under a blanket of N2. The mixture was homogenized witvigorous stirring and heating to about 100° C.
The reaction mixture was then kept at temperature in a range from about 100° C. to about 105° C. and was stirred slowly. To this was added about 1.52 mmol of non-radioactive isotopically enriched iron-57 carbonyl (Fe(CO)5), resulting in instantaneous and aggressive reaction. Non-radioactive Iron-57 enriched carbonyl (Fe(CO)5) addition was completed in less than a minute and the intense reaction subsided. The reaction mixture was then heated to a temperature in a range from about 120° C. to about 130° C. under N2 and maintained at temperature for 1 hour with vigorous stirring.
About 0.76 mmol of non-radioactive isotopically enriched iron-57 carbonyl (57Fe(CO)5) was then added to the reaction mixture. The temperature of the reaction mixture was rapidly increased to about 300° C. to allow the reaction mixture to reflux. After 1 hour of refluxing and stirring at about 300° C., the color of the reaction mixture turned black. The reaction mixture was then cooled to room temperature, and an equal volume amount of isopropyl alcohol was added to the reaction mixture, yielding a black precipitate. The precipitate was separated by centrifuging and collected by magnetic decantation. Particles were readily dispersed in toluene and octane, forming homogeneous solutions. Crystal structure, composition, and particle size analysis of the powder were obtained by transmission electron microscopy (TEM) imaging, energy dispersive x-ray (EDX) elemental analysis, x-ray absorption spectroscopy (XAS), and selected area electron diffraction/x-ray diffraction (SAED-XRD) crystal symmetry pattern indexing. The powder obtained was found to comprise monodisperse mixed non-radioactive isotopically enriched γ-iron-57 oxide/ferrite γ-(Fe2O3)1-y(Fe3O4)y nanocrystals, each having a particle size of about 5 nm(±)0.5 nm. The y-value is generally about 0.4 to about 0.5.
Surface treatment of the non-radioactive traceable metal isotope enriched SPIO nanoparticles is required prior to mammalian injection. To treat the surface, one example treatment may be comprised of: adding tetrahydrofuran (10 mL) to a vial containing 3.25 mg Fe/mL 5 nm SPIO in tetrahydrofuran (4.0 mL, 13 mg Fe, 0.232 mmol), followed by 50% PEI silane in isopropyl alcohol (2.0 mL). The resulting cloudy solution was sonicated for 2 hours. Isopropanol (4.0 mL) was then added and sonicated for an additional 16 hours. Concentrated NH4OH (1.0 mL, 14.8 mmol) was then added and stirred at room temperature for 4 hours. The solution was then diluted with H2O (10 mL) and extracted with hexanes (3.times.10 mL) and ethyl acetate (3.times.10 mL). Any remaining organics in the aqueous layer were removed in vacuo. The resulting homogeneous aqueous solution was passed through a 200 nm syringe filter followed by a 100 nm syringe filter. The solution was then diluted with H2O (10 mL total volume) and purified using a 100 kDa MW cutoff filter (2680.times.g until .about.3 mL of solution remained). The centrifuge filtration process was preferably carried out a total of six times. The final pH of the solution was adjusted to about 7.4 to about 7.7 using concentrated HCl as necessary. Treated in this way, the non-radioactive traceable metal isotope enriched SPIO nanoparticles may be used for mammalian injection.
Example 3
Method for Sample Collection
Non-radioactive isotope-enriched superparamagnetic iron oxide (57Fe labeled SPIO, referred as 57FeSPIO) nanoparticles were injected into rats (female Lewis rats,  ̃200 g) and mice (female Swiss Webster mice,  ̃25 g), and blood clearance and tissue distribution were determined. Blood was collected either by catheterization of the tail vein or by terminal cardiac puncture of the rodents. Blood collection tubes were treated with anticoagulant (to achieve 10 U/mL heparin or 5 mM EDTA). Tubes for tissue collection were pre-weighed for determining the total organ weight after collection of sample.
Blood samples were collected from the rats and mice before injection of isotope-enriched superparamagnetic iron oxide (57FeSPIO) agent. The animals were then injected with a known quantity (typically chosen to be between 1 to 5 mg Fe/kg body weight) of 57FeSPIO nanoparticles through the tail vein. Blood samples ( ̃50 uL) were collected, with typical time points at 5 & 30 min, and 1, 2, 4, 8, 24 and 48 hours following injection. To collect plasma sample, the blood sample was immediately placed on ice and centrifuged cold (e.g., 300×g, 10 min, at 4° C.).
At the terminal time point, typically 1, 7 or 14 days post injection, blood samples were collected by cardiac puncture under deep anesthesia. The animal was euthanized and tissues of interest were collected, rinsed in saline and weighed.
Example 4
MR Imaging
In addition to the collection of blood, plasma and tissue samples described above, magnetic resonance (MR) images were also collected at time points of interest. Images were typically collected using T1, T2 and T2* weighted pulse sequences. Field strength of 1.5 Tesla; R1 ̃10-20 mM-1 s−1. R2 ̃40-60 mM-1 s−1 were used (on a per iron atom basis). The MR data enabled comparison of the 57Fe labeled SPIO (57FeSPIO) nanoparticle's biodistribution determined analytically with the tissue associated contrast enhancement in the various tissues.
Example 5
Method for Sample Digestion and Determination of Spike Recovery
The blood and tissue samples were stored at −20° or −80° C. prior to elemental analysis. The tissues samples were thawed and coarsely homogenized with a blade. The concentration of 57Fe and the ratio of 57Fe to 54Fe ratio were then determined.
Samples were accurately weighed, and added into Mars 5 microwave vessels. 2.5 ml of HNO3, 1 ml of H2O2 and 1.5 ml H2O were added to each microwave vessel. The samples were allowed to stand overnight to pre-digest. The samples were digested in a Mars 5 microwave digestion system. The vessels were allowed to cool. The solutions were quantitatively transferred to 50 mL disposable plastic centrifuge tubes and brought up to 20 gm. Similarly, blanks and spiked samples were prepared and analyzed. For the spiked samples, known amount of 57Fe was added to an aliquot of the biological specimen before the microwave digestion started and was prepared and analyzed in the same fashion as the samples. Spike recovery was then calculated according the following equation:




  
    Spike
    ⁢
    
      
    
    ⁢
    recovery
  
  =
  
    
      
        
          
            
              
                
                  
                     
                    57
                  
                  ⁢
                  Fe
                
                ⁢
                
                  
                
                ⁢
                in
                ⁢
                
                  
                
                ⁢
                the
                ⁢
                
                  
                
                ⁢
                spiked
                ⁢
                
                  
                
                ⁢
                sample
              
              -
            
          
        
        
          
            
              
                
                   
                  57
                
                ⁢
                Fe
              
              ⁢
              
                
              
              ⁢
              in
              ⁢
              
                
              
              ⁢
              the
              ⁢
              
                
              
              ⁢
              unspiked
              ⁢
              
                
              
              ⁢
              sample
            
          
        
      
      
        
          
            
              
                
                  
                     
                    57
                  
                  ⁢
                  Fe
                
                ⁢
                
                  
                
                ⁢
                that
                ⁢
                
                  
                
                ⁢
                was
                ⁢
                
                  
                
                ⁢
                added
                ⁢
                
                  
                
                ⁢
                to
              
            
          
          
            
              
                the
                ⁢
                
                  
                
                ⁢
                spiked
                ⁢
                
                  
                
                ⁢
                sample
                ⁢
                
                  
                
                ⁢
                
                  (
                  known
                  )
                
              
            
          
        
        ⁢
        
          
        
      
    
    ×
    100
  




The spike recovery was used as the quality assurance check to verify the accuracy of the analytical results. Spike recoveries typically ranged from 90-110%. This indicated that all 57Fe analyte has been fully digested and accounted for the analysis with good accuracy. The 57Fe enrichment in the digested samples was determined by elemental analysis using an Element 2 ICP-MS instrument as described below.
Example 6
Elemental Analysis: Solution Nebulization ICP-MS (Thermo Finnegan, Element 2)
All digested samples were diluted 40 times for ICP-MS analysis with the addition of 10 ppb 89Yttrium (89Y) as an internal standard. The analyte concentrations were calculated by comparison with a known series of matrix matched external calibration standards (0-50 ppb), containing 10 ppb 89Y as internal standard. A 200 ppb Fe standard solution (with natural abundance) prepared from High Purity Standard (Charlston, S.C. 29423, USA) stock solution was analyzed to correct for the difference between the natural abundance and the measured ratio. The same solution was also analyzed after every 5 samples to check and correct for the drift of 57Fe/54Fe ratio. All samples were analyzed on the following instrumentation, using the conditions outlined in Table 1:







TABLE 1





Instrumental parameters for the ICP-MS analysis

















Instrumentation



Element 2



Elemental Scientific E2 Autosampler



Poly-nebulizer from Elemental Scientific (self aspirating)



Finnegan E2 torch with sapphire insert



Method Conditions



Pump Rate: self aspirating



Detector: SEM










Auto Masslock: On
Sample Uptake: 30 sec



ICP RF Power: 1050 watts
Rinse Time: 15 sec















Masses
Fe57
Fe54







Run
3
3



Pass
3
3



Resolution
Medium (4000)
Medium (4000)










10 ppb of isotope 89Y was added to both the standards and all the samples. Isotope 89Y was used as internal standard where 57Fe and 54Fe intensities were normalized to 89Y to compensate for any instrument drift in a long run and also to correct for any potential matrix impact.
A schematic illustrating the data obtained by elemental analysis of digested tissues is shown in FIG. 1. A non-radioactive traceable iron isotope enriched SPIO nanoparticle (57FeSPIO) may be employed by the present technique to determine 57FeSPIO concentration in biological samples for characterization of biodistribution and clearance of the nanoparticle. As illustrated, there were 3 isotopic ratios of 57Fe to 54Fe that were relevant in this analysis. One of the three ratios was the ratio of non-radioactive traceable iron isotopes (57FeSPIO to 54FeSPIO) in the superparamagnetic iron oxide (57FeSPIO) nanoparticle 10 (generally represented by ‘S’), as shown in FIG. 1. As seen in FIG. 1 a nanoparticle 10 comprises 57Fe and other isotopes of Iron (for example 56Fe, 54Fe, 58Fe or combinations thereof). The ratio of non-radioactive traceable iron isotopes (57Fe to 54Fe) was measured by elemental analysis for each of the 57FeSPIO. It should be noted that, the ratio S is generally known for a given non-radioactive traceable 57Fe enriched superparamagnetic iron oxide (57FeSPIO) nanoparticle preparation. Second of the three ratios is the ratio of natural abundance (endogenous Iron content of the tissue) of the above-mentioned isotopes (57Fenatural to 54Fenatural), in a tissue sample 12 (generally represented by ‘N’). As will be appreciated by those skilled in the art, the value of N is generally a known quantity. The third ratio is the ratio of the non-radioactive traceable iron isotopes (57Fesample to 54Fesample) (generally represented by ‘M’) measured in a given tissue sample 14 by elemental analysis upon the tissue sample being injected with 57FeSPIO. The nanoparticle enriched tissue sample 14 contains 57Fe enriched SPIO nanoparticles as well as the endogenous Iron isotopes already present in the tissue sample. Note that a similar analysis could be implemented using total iron or another iron isotope in place of 54Fe in these ratios, and likewise other isotopes could be used for labeling by enrichment.
M depends on the both on the endogenous iron content of the tissue and on the concentration of 57FeSPIO in the tissue:




  
    
      
        M
        =
        
        ⁢
        
          
            Fe
            sample
            
            
              
              57
          
          
            Fe
            sample
            
            
              
              54
          
        
      
    
  
  
    
      
        =
        
        ⁢
        
          
            
              Fe
              SPIO
              
              
                
                57
            
            +
            
              Fe
              natural
              
              
                
                57
            
          
          
            
              Fe
              SPIO
              
              
                
                54
            
            +
            
              Fe
              natural
              
              
                
                54
            
          
        
      
    
  




The concentration of 57Fe in the sample attributable to 57FeSPIO can be calculated as:




  
    Fe
    SPIO
    
    
      
      57
  
  =
  
    
      
        
          Fe
          sample
          
          
            
            57
        
        /
        M
      
      -
      
        
          Fe
          sample
          
          
            
            57
        
        /
        N
      
    
    
      
        1
        /
        S
      
      -
      
        1
        /
        N
      
    
  




Knowing the 57Fe enrichment in the 57FeSPIO preparation (e.g., 57FeSPIO: SPIO (SPIO with natural Fe isotope), determined for each core prep), the total iron concentration in the tissue attributable to 57FeSPIO is given by:

SPIO=57FeSPIO/Enrichment








TABLE 2







Natural abundance of common iron isotopes.










Isotope
Natural Abundance














54Fe
5.8%



56Fe
91.7%



57Fe
2.2%



58Fe
0.3%










Natural abundance of common iron isotopes are given in Table 2. The iron concentration in tissue attributable to SPIO and 57FeSPIO may be used to calculate percentage of injected dose (% ID or % ID/g) statistics by dividing by the total iron dose of SPIO.
While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

## Claims

1. A nanoparticle, comprising:
at least one inorganic core; and
at least one coating substantially covering the inorganic core, wherein the inorganic core comprises at least one enriched non-radioactive traceable metal isotope wherein the at least one of the enriched non-radioactive traceable element comprises 57Fe.

2. The nanoparticle of claim 1, wherein the nanoparticle has a diameter in a range of from about 1 nm to about 1000 nm.

3. The nanoparticle of claim 1, wherein the nanoparticle has a diameter in a range of from about 1 nm to about 10 nm.

4. The nanoparticle of claim 1, wherein the enriched non-radioactive traceable metal isotope is present in a range of from about 1% to about 99%.

5. The nanoparticle of claim 1, wherein the inorganic core further comprises at least one natural metal isotope of a metal selected from a group consisting of iron, manganese, copper, titanium, cadmium, cobalt, nickel, chromium, gadolinium, yttrium, zinc, molybdenum, vanadium, and derivatives thereof.

6. The nanoparticle of claim 1, wherein the enriched non-radioactive traceable metal isotope further comprises at least one isotope of a metal selected from a group consisting of iron, manganese, copper, titanium, cadmium, cobalt, nickel, chromium, gadolinium, yttrium, zinc, molybdenum, vanadium, and combinations thereof.

7. The nanoparticle of claim 1, wherein the enriched non-radioactive traceable metal isotope further comprises at least one Fe isotope selected from a group consisting of 54Fe, and 58Fe.

8. The nanoparticle of claim 1, wherein the coating comprises a biocompatible material.

9. The nanoparticle of claim 8, wherein the biocompatible material comprises an organo-silane compound.

10. The nanoparticle of claim 9, wherein the organo-silane compound is further derivatized with polyethylene glycol, polyethylene imine, dextran, or combinations thereof.

11. A superparamagnetic nanoparticle comprising:
at least one superparamagnetic iron oxide core, and at least one coating substantially covering the superparamagnetic iron oxide core, wherein the superparamagnetic iron oxide core comprises at least one enriched non-radioactive traceable metal isotope wherein the at least one of the enriched non-radioactive traceable element comprises 57Fe; and
the superparamagnetic nanoparticle has a diameter in a range of from about 1 nm to about 1000 nm.

12. The superparamagnetic nanoparticle of claim 11, wherein the enriched non-radioactive traceable metal isotope further comprises at least one Fe isotope selected from a group consisting of 54Fe, and 58Fe.

13. The superparamagnetic nanoparticle of claim 11, wherein the superparamagnetic nanoparticle has a diameter in a range of from about 1 nm to about 10 nm.

14. The superparamagnetic nanoparticle of claim 11, wherein the coating comprises a biocompatible organo-silane compound.

15. A method of forming a plurality of nanoparticles, wherein the method comprises
(a) combining a non-polar aprotic organic solvent, an oxidant, and a first surfactant, to form a first solution;
(b) adding at least one organometallic compound to the first solution, to form a second solution, wherein the at least one organometallic compound comprises at least one enriched non-radioactive traceable metal isotope and at least one ligand wherein the at least one of the enriched non-radioactive traceable element comprises 57Fe; and
(c) heating the second solution to a temperature in a range of from about 30° C. to about 400° C., to form the plurality of nanoparticles, wherein the plurality of nanoparticles comprise at least one inorganic core and at least one coating substantially covering the inorganic core, wherein the inorganic core comprises at least one enriched non-radioactive traceable metal isotope.

16. The method of claim 15, wherein the organometallic compound further comprises at least one enriched non-radioactive traceable metal isotope selected from a group consisting of 54Fe, and 58Fe.

17. A method for determining a distribution of nanoparticles in a biological material comprising the steps of:
(a) introducing the nanoparticles into the biological material,
wherein the nanoparticles comprise at least one inorganic core, wherein the inorganic core comprises at least two metal isotopes in a predetermined ratio, wherein at least one metal isotope is enriched non-radioactive traceable metal isotope wherein the at least one of the enriched non-radioactive traceable element comprises 57Fe; and
(b) determining the distribution of the nanoparticles in the biological material based on the predetermined ratio of the metal isotopes.

18. The method of claim 17, wherein the enriched non-radioactive traceable metal isotope further comprises at least one isotope of a metal selected from a group consisting of iron, manganese, copper, titanium, cadmium, cobalt, nickel, chromium, gadolinium, yttrium, zinc, molybdenum, vanadium, and combinations thereof.

19. The method of claim 17, wherein the inorganic core further comprises 54Fe or 58Fe.

20. The method of claim 17, wherein the nanoparticle has a diameter in a range of from about 1 nm to 1000 nm.

21. The nanoparticle of claim 17, wherein the nanoparticle has a diameter in a range of from about 1 nm to about 10 nm.

22. The method of claim 17, wherein the enriched non-radioactive traceable metal isotope is present in a range of from about 1% to about 99%.