WO2011111024A2

WO2011111024A2 - A method of magnetic resonance imaging for detecting calcium

Info

Publication number: WO2011111024A2
Application number: PCT/IB2011/051019
Authority: WO
Inventors: Yoram Cohen; Amnon Bar-Shir; Liat Avram-Biton
Original assignee: Ramot At Tel-Aviv University Ltd.
Priority date: 2010-03-10
Filing date: 2011-03-10
Publication date: 2011-09-15
Also published as: WO2011111024A3

Abstract

Methods and compositions for magnetic resonance spectroscopy (e.g., imaging) of calcium ions in samples such as physiologic media and tissues both in vitro and in vivo, utilizing superparamagnetic iron oxide nanoparticles coated with an alginate, are provided. The superparamagnetic iron oxide nanoparticles coated with an alginate form aggregates with calcium ions, and the formation and/or level of such aggregates are indicative for the presence and/or level, respectively, of calcium ions in the sample. The methods and compositions can be used for monitoring a presence and/or progression of a medical condition associated with calcium ions.

Description

A METHOD OF MAGNETIC RESONANCE IMAGING FOR DETECTING

CALCIUM

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to diagnosis, and more particularly, but not exclusively, to a method and a composition for detecting calcium ions in aqueous samples such as physiologic environments, and for monitoring medical conditions associated with a change in a level of calcium ions.

Metals ions are essential for sustaining living cells which maintain different ion concentration of metals in their intra- or extra-cellular spaces. Ca²⁺, for example, is kept at low concentrations (below 10^~4 M) in the cytosol and at high concentrations (more than 10^~3 M) in the extracellular space. Such calcium homeostasis, which is essential for the survival and function of live organism, is controlled by complex regulatory systems [Domaille et al. Nat. Chem. Biol. 4, 168-175 (2008)].

Changes in intracellular Ca²⁺ concentrations initiate a cascade of signaling effects that can regulate many different cellular functions. Ca²⁺ plays an important role in many biological processes such as fertilization, cell differentiation, cell proliferation, activation of transcription factors, apoptosis, cardiac contractility and neurotransmission [Hofer, A. M. & Brown E. M. Nat. Rev. Mol. Cell Biol. 4, 530-538 (2003)]. Extracellular Ca²⁺ is also essential for biological processes such as blood clotting, the maintenance of tight junction in epithelial sheets and skeletal remodeling [Berridge et al. Nat. Rev. Mol. Cell Biol. 1, 1 1-21 (2000)].

Currently, the technologies that are used for Ca²⁺ imaging are mostly based on fluorescent methodologies. These methodologies, however, suffer from several limitations including, inter alia, low tissue penetration and small localized field of view.

Magnetic resonance imaging (MRI) is a method which utilizes the nuclear magnetic resonance (NMR) phenomenon for finding out the local distributions of nuclei density and nucleus-related NMR properties of an object or the physical and chemical characteristics having an effect thereon. Such NMR properties include mainly longitudinal relaxation (characterized by longitudinal relaxation time Tj), transverse relaxation (characterized by transverse relaxation time T₂), relaxation in a rotating frame of reference (characterized by relaxation time Tip), transverse relaxation in the presence of field inhomogeneity (characterized by relaxation time T₂*), chemical shift and coupling factors between the nuclei. NMR properties are also affected by different physical phenomena, such as diffusion, perfusion, paramagnetic materials, ferromagnetic materials, viscosity and temperature, etc.

Magnetic resonance imaging (MRI) is currently considered one of the most important imaging modalities in biomedicine. The advantages of the MRI methodology lie on its non-invasiveness, its high spatial and temporal resolutions and its unlimited penetration to biological tissues. The image contrast in MRI may be based on a host of intrinsic physical parameters, including the nuclear spin density, relaxation times and molecular motions. MRI images are essentially images of water protons or physical parameters of those protons. However, MRI is not specific to particular molecules or biological processes.

In some situations it is not possible to generate enough image contrast to adequately show the anatomy or pathology of interest by adjusting the imaging parameters alone, and therefore a contrast agent needs to be administered. Most contrast agents used in MRI are selected for their specific magnetic properties, and more recently, superparamagnetic contrast agents, e.g., iron oxide nanoparticles, have become commercially available and approved for medicinal use. These agents cause water to appear very dark on T₂- or T₂*- weighted MR images and may be used, for example, for liver imaging, as normal liver tissue retains the agent, but abnormal areas (e.g., scars, tumors) do not.

Superparamagnetic iron oxide nanoparticles (SPIO-NPs) with appropriate surface chemistry have been widely used for applications such as magnetic resonance imaging contrast enhancement [Wang et al., 2001, Eur. Radiol., 11, 2319-2331]. Some SPIO-NPs contrast agents can also be taken orally, to improve visualization of the gastrointestinal tract, and to prevent water in the gastrointestinal tract from obscuring other organs (e.g., the pancreas).

Recently, some progression has been achieved in the design of unique "smart" probes for MRI, which enable noninvasive assessment of essential biological processes and pathologies [Louie et al. Nat. Biotechnology 18, 321-325 (2000); Lee et al. Nat. Med. 13, 95 - 99 (2007)]. A few MRI probes were developed for the purpose of Ca²⁺ MR imaging, based on the induction of change of Tj (positive contrast agent) or T₂ relaxation times of water in the presence of Ca²⁺. Most of the positive probes are based on functionalized Gd³⁺ complexes, which increase water signal in Ti weighted MR images in the presence of Ca²⁺ ions [see, Li et al. J. Am. Chem. Soc. 121, 1413-1414 (1999); Li et al. J. Inorg. Chem. 41, 4018-4024 (2002); Dhingra et al. Chem. Comm. 29, 3444-3446 (2008); Angelovski et al. ChemBioChem 9, 1729-1734 (2008); Mishra et al. Inorg. Chem. 47, 1370-1381 (2008); Dhingra et al. J. Biol. Inorg. Chem. 13, 35-46 (2008)]. Manganese enhanced MRI (MEMRI) has also been suggested as an imaging methodology for qualitatively monitoring Ca²⁺ influx with the aid of paramagnetic Mn²⁺ [Lin, Y. J. & Koretsky, A. P. Magn. Reson. Med. 38, 378-388 (1997)].

On the other hand, negative MRI contrast agents that are based on SPIO-NPs, are expected to change ^-weighted MR images as a result of nanoparticles aggregation in the presence of Ca²⁺. Atanasijevic et al. developed MR Ca²⁺ indicators based on SPIO-NPs functionalized with the calcium-sensing protein, calmodulin, and its target peptides [Atanasijevic et al. Proc. Nat. Acad. Sci. U.S.A. 103, 14707-14712 (2006); Atanasijevic, T. & Jasanoff, A. Nat. Protocols 2, 2582-2589 (2007)]. These probes were found to increase T₂, yet, were found to be limited to detection of intracellular Ca²⁺ at concentrations near 1 μΜ and below due to saturation of the system at higher calcium concentrations. Taktak et al. also developed functionalized SPIO-NPs sensor for Ca²⁺ MR imaging [Taktak et al., Langmuir 24, 7596-7598 (2008)].

Alginates are linear copolymers consisting of β-D-mannuronic (M) and a-L- glucuronic (G) acid residues. It has been demonstrated that Ca²⁺ ions are able to complex G blocks from two different chains of alginate polysaccharides, forming an "egg-box" model [Fang et al., J. Phys. Chem. B 111, 2456-2462 (2007)]. The cross- linking of alginates results in alginate gel formation which is not obtained in the presence of monovalent (such as Na⁺ or K⁺) or other divalent cations (such as Mg²⁺) [Chen et al, Langmuir 23, 5920-5928 (2007); Chen et al, Environ. Sci. Technol. 40, 1516-1523 (2006)].

MA et al. described the preparation of SPIO-NPs stabilized by alginate coating

[Ma et al., Int. J. Pharm. 333, 177-186 (2007)] and described the pharmacokinetics and the tissue distribution of those iron oxide-based MR contrast agents [Ma et al., Int. J. Pharm. 354, 217-226 (2008)]. In these SPIO-NPs the carboxylate group (COO ) of the alginate and the iron ion interact, thus forming stable alginate-coated SPIO-NPs systems [Laurent et al, Chem. Rev. 108, 2064-2110 (2008)]. SUMMARY OF THE INVENTION

The present inventors have designed and successfully practiced methods and compositions utilizing water-dispersible or suspendable alginate-coated superparamagnetic iron oxide nanoparticles (SPIO-NPs) in NMR/MR spectroscopy routines, as well as in other spectroscopic methodologies, for determining a presence and/or level of calcium ions, and have demonstrated that such alginate-coated SPIO- NPs can be utilized for detecting and imaging different concentrations of Ca²⁺ in aqueous solutions at physiological and non-physiological pH and temperature, as well as in other physiological media such as blood, serum, cell cultures and living organs both in vitro and in vivo.

The alginate-coated SPIO-NPs presented herein are suitable for detecting a wide range of Ca²⁺ concentrations by magnetic resonance imaging or spectroscopy (MRI or MRS), which may be relevant to different regimes of calcium imaging (i.e., following intracellular Ca²⁺, following extracellular Ca²⁺, following blood Ca²⁺ levels, following changes in Ca²⁺ as a result of different pathologies, functional magnetic resonance imaging (fMRI), etc.).

The alginate-coated SPIO-NPs presented herein are also suitable for detecting a wide range of Ca²⁺ concentrations by other spectroscopic methods such as DLS.

The alginate-coated SPIO-NPs presented herein are further suitable for detecting relatively high concentrations of Ca²⁺ by magnetic resonance imaging or spectroscopy (MRI or MRS), by adding and controlling the amount of free alginate in the monitored media.

The platform of calcium detection, according to some embodiments of the present invention, is also suitable for detecting a wide range of Ca²⁺ concentrations in physiologic and other biologic samples which contain many other substances, including other metal ions, which may share some chemical attributes with calcium ions. For example, the presence of the physiologically ubiquitous magnesium in the form of the divalent cation Mg²⁺, and the presence of other metal ions, does not mask the signal obtained for calcium ions using the method presented herein.

Using alginate-coated SPIO-NPs as biomarkers for Ca²⁺ MR imaging or spectroscopy, according to some embodiments of the invention, is highly effective in diagnosing different diseases, in monitoring and studying the functions of healthy and/or diseased tissues and organs such as brain and heart, in understanding complex biological processes, and in the development of new drugs, all of which are associated with changes in calcium ions levels.

Hence, according to an aspect of embodiments of the present invention, there is provided a method of determining a presence and/or a level of calcium ions in a sample, the method comprising:

contacting the sample with a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate; and

subjecting the sample to magnetic resonance spectroscopy (e.g., imaging), to thereby detect a formation of aggregates of at least a portion of the superparamagnetic iron oxide nanoparticles coated with the alginate, wherein a formation and/or level of formation of the aggregates is indicative of the presence and/or level of calcium ions in the sample, thereby determining a presence and/or a level of calcium ions in the sample.

In some embodiments, the method presented herein is for determining a presence and/or level of calcium ions in a range of from 100 μΜ to 5 mM.

In some embodiments, the method further comprises contacting the sample with a free alginate.

In some embodiments, the concentration of the free alginate ranges from 0.02 to 0.06 percent by weight of the total weight of the sample, and the method presented herein is for determining a presence and/or level of calcium ions in a range of from 2 mM to 5 mM.

In some embodiments, the concentration of the free alginate ranges from 0.002 to 0.02 percent by weight of the total weight of the sample, and the method presented herein is for determining a presence and/or level of calcium ions in a range of from 0.2 mM to 2 mM.

In some embodiments, the method presented herein is for determining a presence and/or a level of calcium ions in the presence of ions of a metal other than calcium. In some embodiments, the method presented herein is for determining a presence and/or a level of calcium ions in the presence of magnesium ions (e.g., Mg⁺²).

According to another aspect of embodiments of the present invention, there is provided a composition which includes a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate, the composition is identified for use in determining a presence and/or a level of calcium ions in an aqueous medium by magnetic resonance spectroscopy (e.g., imaging) of a sample.

According to another aspect of embodiments of the present invention, there is provided a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate, for use in determining a presence and/or a level of calcium ions in an aqueous medium by magnetic resonance spectroscopy (e.g., imaging) of a sample.

According to another aspect of embodiments of the present invention, there is provided a use of a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate in the manufacture of a diagnostic agent for determining a presence and/or a level of calcium ions in a sample by magnetic resonance spectroscopy (e.g., imaging).

In some embodiments of the composition, the plurality of superparamagnetic iron oxide nanoparticles coated with an alginate or the use presented herein, is for determining a presence and/or level of calcium ions having a concentration at a range from 100 μΜ to 5 mM.

In some embodiments, the composition further includes a free alginate.

In some embodiments of the plurality of superparamagnetic iron oxide nanoparticles coated with an alginate or of the use presented herein, the alginate-coated SPIO-NPs are used in combination with a free alginate.

In some embodiments of the composition, the plurality of alginate-coated SPIO- NPs or of the use presented herein, the concentration of the free alginate ranges from 0.02 to 0.06 percent by weight of the total weight of the sample, and the magnetic resonance spectroscopy (e.g., imaging) is used for determining a presence and/or level of calcium ions in a range of from 2 mM to 5 mM.

In some embodiments of the composition, the plurality of alginate-coated SPIO- NPs or the use presented herein, the concentration of the free alginate ranges from 0.002 to 0.02 percent by weight of the total weight of the sample, and the magnetic resonance spectroscopy (e.g., imaging) is used for determining a presence and/or level of calcium ions in a range of from 0.2 mM to 2 mM.

In some embodiments of the method, composition, the plurality of alginate- coated SPIO-NPs or the use presented herein, the sample comprises an aqueous medium.

In some embodiments of the method, composition, the plurality of alginate- coated SPIO-NPs or the use presented herein, the sample is selected from the group consisting of an aqueous solution, a biological sample, a bodily fluid sample, a cell culture, a plant sample, a tissue sample, an organ sample and a bodily site.

In some embodiments of the composition, the plurality of alginate-coated SPIO-

NPs or the use presented herein, the detecting is effected is the presence of ions of a metal other than calcium.

According to another aspect of embodiments of the present invention, there is provided a method of monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject, the method comprising: administering to the subject a diagnostically effective amount of a plurality of alginate-coated SPIO-NPs;

subjecting the subject to magnetic resonance spectroscopy (e.g., imaging);

and detecting a formation of aggregates of at least a portion of the alginate - coated SPIO-NPs, wherein a formation and/or level of formation of the aggregates is indicative of the presence and/or level of calcium ions in an organ or tissue of the subject,

thereby monitoring the presence and/or progress of the medical condition.

According to another aspect of embodiments of the present invention, there is provided a use of a plurality of alginate-coated SPIO-NPs in the manufacture of a diagnostic agent for monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject by magnetic resonance spectroscopy (e.g., imaging).

According to another aspect of embodiments of the present invention, there is provided a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate for use in monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject by magnetic resonance spectroscopy (e.g., imaging).

According to another aspect of embodiments of the present invention, there is provided a pharmaceutical composition which includes a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate, the composition is identified for use in monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject by magnetic resonance spectroscopy (e.g., imaging).

In some embodiments of the method, use or composition presented herein, the medical condition is selected from the group consisting of hypocalcaemia, hypercalcaemia, constipation, eating disorders, chronic renal failure, severe acute hyperphosphatemia, ineffective PTH syndroms, pancreatitis, alkalosis, bone pain, kidney stones, psychotic behavior, depression, confusion, psychiatric overtones, cerebral ischemia, cardiac ischemia, cardiac arrest, heart failure, cancer, neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson, multiple sclerosis and Alzheimer disease.

In some embodiments of the diagnostic method, use or composition presented herein, the level of calcium ions in a bodily site of the subject ranges from 100 μΜ to 5 mM.

In some embodiments, the diagnostic method presented herein further includes administering to the subject an effective amount of a free alginate.

In some embodiment of the diagnostic use presented herein, the diagnostic agent is used in combination with a free alginate.

In some embodiment, the plurality of superparamagnetic iron oxide nanoparticles coated with an alginate for diagnostic use, is used in combination with a free alginate.

In some embodiment, the diagnostic composition presented herein further includes a free alginate.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings and images. With specific reference now to the drawings and images in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings and images makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 presents a schematic illustration of the effect of adding free alginate to a system having alginate-coated SPIO-NPs and calcium ions, suggesting that the free alginate and the calcium ions bridge between alginate-coated SPIO-NPs to form larger aggregates, which grow larger with the growing concentration of free alginate and the calcium ions;

FIGs. 2A-D present various modes of expressing the effect of Ca²⁺ on alginate- coated SPIO-NPs, wherein the visual inspection of the alginate-coated SPIO-NPs 3 hours after the addition of H₂0, 0.5 mM/1.0 mM of Ca²⁺ or Mg²⁺, reveals clear precipitation only in the 1 mM Ca²⁺ solution (FIG. 2A), the particle's size analyses of alginate-coated SPIO-NPs 3 hours after the addition of H₂0, 0.5 mM Ca²⁺ or 0.5 mM Mg²⁺ as obtained from DLS measurements, reveal a clear increase in the size of the alginate-coated SPIO-NPs in the presence of 0.5 mM Ca²⁺ (FIG. 2B), r₂-weighted MR images (TR/TE=5000/20 ms) of aqueous solutions of alginate-coated SPIO-NPs with 0.002 % of free alginate in the absence and in the presence of 0.5 mM/1.0 mM of Ca2+ or Mg²⁺, show that Ca²⁺ has dramatic effect on the contrast, while Mg²⁺ has practically no effect on the signal intensity (FIG. 2C), and in the bar plot of percentage change in the MR signal intensity, of the ₂-weighted MR images shown in FIG. 2C, as a function of the reference sample (FIG. 2D); FIG. 3 presents ₂-weighted MR images (TR/TE=5000/ 10ms) of aqueous solution containing alginate-coated SPIO-NPs and the indicated Ca²⁺ concentrations (in mM), acquired 4 hours after the addition of Ca²⁺ to different alginate-coated SPIO-NPs solutions/suspensions which are placed in the matrix by rows, wherein row "a" is a solution without free alginate, row "b" is a solution with 0.002 % of 14 kDa alginate, row "c" is a solution with 0.002 % 30 kDa alginate, row "d" is a solution with 0.02 % of 14 kDa alginate, and row "e" is a solution with 0.02 % 30 kDa alginate;

FIGs. 4A-E present comparative plots of the percentage of signal intensity change in the ^-weighted MR images (TR/TE=5000/20ms) 3-5 hours after the addition of the indicated concentrations of Ca²⁺ and Mg²⁺ ions to alginate-coated SPIO-NPs solutions, in the presence of different amount of 30 kDa and 14 kDa alginates, wherein no addition of free alginate is shown in FIG. 4A, addition of 0.002 % of 30 kDa alginate is shown in FIG. 4B, with 0.02 % 30 kDa alginate is shown in FIG. 4C, with 0.002 % of 14 kDa alginate is shown in FIG. 4D, with 0.02 % 14 kDa alginate is shown in FIG. 4E;

FIGs. 5A-C present ₂-weighted MR images (TR/TE=5000/20ms) accompanied with a plot of the percentage change in the MR signal intensity of the water in aqueous solution of the alginate-coated SPIO-NPs in the presence of 0.5 mM Mg²⁺ after the addition of 0.002 % 30 kDa alginate (FIG. 5A), after addition of 0.02 % 30 kDa alginate (FIG. 5B), and after addition of 0.02 % 30 kDa alginate 0.1 mM changes in Ca²⁺ levels (FIG. 5C);

FIGs. 6A-B present r₂-weighted MR images (TR/TE=5000/ 10ms (FIG. 6A) and 5000/20ms (FIG. 6B)), 4 hours after the addition of different concentration of Ca²⁺/Mg²⁺ (0-4.0 mM) to aqueous solution of alginate-coated SPIO-NPs to which 0.05 % of 30 kDa alginate was added, showing the ability to detect very high concentration of calcium;

FIGs. 7A-B present comparative bar-plots of the percentage change in the signal intensity in ₂-weighted MR images (TR/TE=3000/20ms), 45 minutes after addition of different concentration of Ca²⁺ to fetal bovine serum (FBS) solutions containing alginate-coated SPIO-NPs (FIG. 7A) and to Dulbecco's Modified Eagle Medium (DMEM) solutions containing alginate-coated SPIO-NPs (FIG. 7B), wherein all samples had 0.02 % of 30 kDa alginate added thereto, showing that the sensitivity to different concentration of calcium exists even in biological media; FIG. 8 presents a bar-plot of the percentage change in the signal intensity in T₂- weighted MR images (TR/TE=3000/40ms) of solutions with and without cells, 30 minutes after addition of alginate-coated SPIO-NPs with 0.002 % (bars "a" and "b") or 0.02 % (bars "c" and "d") of 30 kDa alginate to DMEM (bars "a" and "c") or ischemic DMEM (bars "b" and "d"), showing that in ischemic DMEM there is a release of calcium which can be detected by our MRI probe, and showing that the sensitivity to different concentration of calcium exists even in biological media (data obtained from 11 different experiments, n=l 1);

FIG. 9 presents a comparative bar-plot of the percentage of cells survival at 4, 8 and 24 hours after exposure of C8-D1A astrocyte cells to HEPES buffer 10 mM as control and to SPIO-alginate NP diluted 1 : 1 with HEPES buffer 20 mM, showing that no toxic effect of the alginate-coated NPs on these cells was found;

FIGs. 10A-B present two in vivo ₂-weighted MR images (TR/TE=3500/60ms) of two rats, seven days after the injection of quinolinic acid (150 nmol of quinolinic acid in 1 μΐ of PBS) to the right hemisphere, and 1 μΐ of PBS to the left hemisphere, and a few hours after the bilateral injection of 2 μΐ of the aqueous solution of (FIG. 10A) alginate-coated SPIO-NPs, and the bilateral injection of FERIDEX® (FIG. 10B), showing a much darker area on the right lobe in FIG. 10A, that matches the quinolinic acid lesion which is shown only in the damaged striatum, in which Ca²⁺ concentration should be higher; and

FIG. 11 presents a comparative bar-plot of hypointense pixels counted in weighted digital images taken from 16 rats injected bilaterally with alginate-coated SPIO-NPs and 8 rats injected bilaterally with FERIDEX®, showing the ratio between the right and left hemispheres injected with QA and PBS respectively.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

As widely known in the art, metal nanoparticles suspended in an aqueous solution affect the magnetic resonance signal of the surrounding water molecules; an effect that is harnessed in magnetic resonance spectroscopy (e.g., imaging) techniques for decades.

As discussed hereinabove, calcium ions are involved in a variety of biological processes and systems, and a change in calcium ions concentration can detrimentally affect these processes and systems. Thus, there has been an increasing need for an imaging platform that would enable the detection of changes in Ca²⁺ levels at a wide concentration range.

Current methodologies for detecting changes in calcium ions levels mostly involve direct and typically destructive metal ion or elemental analysis of a sub-sample, which is extracted from the studied sample at various time intervals. For example, if the sample is a live animal, one animal has to be sacrificed for each time-point (interval) of data, the organ or tissue of interest has to be removed, and a sub-sample extracted therefrom and analyzed for calcium.

Ma et al. (supra, 2008) describe the pharmacokinetics and tissue distribution of alginate-coated SPIO, and investigate its potential in detecting malignancies when used as a magnetic resonance contrast agent, yet have not explored an interaction of alginate- coated SPIO NPs with divalent ions such as calcium ions.

While studying the effect of aggregation of iron particles on the magnetic signal of the surrounding water, the present inventors have found that the signal increases synergistically, namely in a non-linear manner, relative to the number of metal particles in the aggregates. These findings lead the present inventors to explore the possibility of using alginate-coated SPIO-NPs to form aggregates in the presence of Ca²⁺. As a result, the present inventors have surprisingly uncovered that alginate-coated SPIO-NPs can be used as a highly sensitive probe for detecting and determining calcium levels at a wide range of concentrations in physiologic media and/or conditions, using magnetic resonance spectroscopy (e.g., imaging) as well as other spectroscopic techniques such DLS. The present inventors have further showed that the addition of free alginate can be used to widen the range of detectable calcium concentration to relatively high concentrations, using the methods presented herein. In addition it has been shown that the methodology presented herein is valid also in the presence of divalent ions other than calcium ions, such as magnesium ions, which do not mask the signal obtained for calcium ions using the method presented herein.

Thus, a new platform for selective detection and determination of a wide range of calcium concentrations in variable aqueous solutions, biological samples and organs, both in vitro and in vivo, by spectroscopic technologies such as MRI and DLS, based on the use of alginate-coated SPIO-nanoparticles, has been developed and provided herewith.

Hence, according to an aspect of some embodiments of the present invention, there is provided a method of determining a presence and/or a level of calcium ions in a sample. The method is effected by contacting the sample with a plurality of superparamagnetic iron oxide nanoparticles (SPIO-NPs) coated with alginate, with or without the addition of free alginate; and subjecting the sample to magnetic resonance spectroscopy (e.g., imaging), to thereby detect a formation of aggregates of at least a portion of the superparamagnetic iron oxide nanoparticles coated with alginate quantitatively, and thereby determining a presence (qualitative detection) and/or a level (quantitative determination) of calcium ions in the sample. The formation and/or level of formation of the aggregates is indicative of the presence and/or level of calcium ions in the sample.

Magnetic resonance imaging (MRI) is a general term used for techniques based on magnetic resonance spectroscopy (MRS). Magnetic resonance spectroscopy (MRS) is used to measure the levels of different metabolites in body tissues. The MR signal produces a spectrum of resonances that correspond to different molecular arrangements of the isotope being "excited". This signature is used to diagnose certain disorders in a bodily site, for example the brain, and to provide information on biologic possesses occurring at the bodily site. Magnetic resonance spectroscopic imaging (MRSI) combines both magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) methods to produce spatially localized spectra from a bodily site. The spectra in each voxel (three dimensional pixel) contains information about metabolites. Dynamic light scattering (DLS)) is a technique which can be used to determine the size distribution profile of small particles or aggregates in suspension in a solution.

For simplicity, the following relates to magnetic resonance imaging. Yet, it is to be understood that any other magnetic resonance spectroscopy are also contemplated, and in some cases (e.g., aqueous solutions), DLS can also be used.

The term "sample", as used herein, refers to any sample which can be analyzed by MR spectroscopy or MR imaging namely any sample which includes at least 10 % water by weight, at least 20 % water, at least 30 % water, at least 40 % water, and at least 50 % water, by weight. Hence, the term sample represents, for example, an aqueous media sample (aqueous solution or suspension) such as a sample of a bodily fluid (e.g., a blood sample, a urine sample), a biological sample such as a cell culture, a tissue sample, or an organ sample, a plant, a living organism or a bodily site in an organism/animal or a human subject, or and any parts of combinations thereof.

The phrase "superparamagnetic iron oxide nanoparticles", abbreviated SPIO- NPs, describes nanometric particles of iron oxide which exhibit extensive paramagnetism resulting from their chemical composition and physical size. Superparamagnetic iron oxide nanoparticles (SPIO-NPs), exhibiting various surface chemistry, have been used for applications such as magnetic resonance imaging contrast enhancement [Wang et al, 2001, Eur. Radiol, 11, 2319-2331], immunoassays, hyperthermia, magnetic drug delivery, magnetofection, cell separation/cell labeling, etc. Furthermore, some SPIO-NPs preparations have already been approved for clinical use, especially for MR imaging, such as Endorem® (diameter 80-150 nm, Advanced Magnetics) and Resovist® (diameter 60 nm, Schering) for liver/spleen imaging. Functionalized magnetic nanoparticles conjugated with antibodies or receptors including epidermal growth factor receptors (EGFRs), her2/neu and folate have been developed for various studies.

These applications need special surface coating of the magnetic particles, which has to be not only non-toxic and biocompatible but also allow targetable delivery with particle localization in a specific well as high quality of the magnetic particles, their size distribution, their shape and surface. The nature of surface coatings and their subsequent geometric arrangement on nanoparticles determine not only the overall size of the resulting colloid but also play a role in the bio-kinetics and bio-distribution of nanoparticles in the body. Some factors related to the clearance of the iron oxide nanoparticles from blood include particle size, dose, surface charge, coating material, stability in physiological environment, and mode of administration. For example, it was shown that the larger the particles, the shorter their plasma half-life. For some iron oxide nanoparticles, such as AMI-25 type nanoparticles exhibiting a diameter of 80-150 nm, the blood half-life is only 6 minutes and approximately 80 % of the injected dose accumulated in the liver and 5-10 % in the spleen within minutes of administration. In comparison, the vascular half-life of NCI 0050 type nanoparticles with diameter of 20 nm is up to 3-4 hours. It is also known that the higher the surface charge, the shorter the residence time of SPIO-NPs in the circulation.

Alginate-coated SPIO-NPs is a composite material in which SPIO-NPs are coated with alginic acid-derived gums. Alginate-coated SPIO-NPs were initially developed to alter the bio-kinetics and bio-distribution of nanoparticles in the body, by altering the surface chemistry of the SPIO-NPs. As alginate is a highly charged polysaccharide having the capacity to absorb water and form insoluble hydrogels, modifying the surface of SPIO-NPs with alginate improves bio-kinetics and bio- distribution of the coated SPIO-NPs.

The term "alginate", as used herein, refers to alginic acid, which is also called algin or alginate, and which is a ubiquitous viscous gum found in the cell walls of brown algae. This term encompasses derivatives and analogs of alginic acid. Alginates are widely used as food additives and elsewhere as coating agents, emulsifiers and gelling agents, as well as in indigestion tablets and in the preparation of dental impressions. The structure of alginate is typically a linear copolymer with homopolymeric blocks of (1-4)- linked β-D-mannuronate (known as the "M unit" or M) and its C-5 epimer a-L- glucuronate (known as the "G unit" or G) residues, respectively, covalently linked together in different sequences or sequence blocks. Alginate oligomers can take the form of homopolymeric blocks of consecutive G-residues known as G-blocks, consecutive M-residues (M-blocks), alternating M and G-residues (MG-blocks), or randomly organized blocks.

In the context of the present embodiments, the term "alginate" encompasses sodium alginate, the sodium salt of alginic acid, and other pharmaceutically acceptable salts, hydrates, analogues and derivatives of alginic acid, as well as alginates of various combinations of M/G unit ratios, all of which are defined as capable of interacting with calcium and alginate-coated SPIO-NPs to form aggregates thereof.

Alginate varieties are typically extracted from seaweed or produced by bacterial genera such as Pseudomonas and Azotobacter. Bacterial alginates are particularly useful for the production of micro- or nanostructures suitable for medical applications, such as embodiments of the present invention. Hence, alginates which are useful in the context of the present invention may be obtained from natural sources such as marine plants and bacteria (by e.g., extraction), or from synthetic processes which further process the naturally occurring alginates. Optionally, the alginates can be synthetically prepared alginates.

It is to be understood that the term "alginate" is meant to encompass alginates of a wide range of viscosity average molecular weights. According to some embodiments of the present invention, the molecular weight of the alginate ranges from 1 Kilo Dalton (kDa) to 1000 kDa, from 1 kDa to 500 kDa, from 1 kDa to 100 kDa, from 50 kDa to 200 kDa, or from 2 kDa to 50 kDa.

The general definition of alginate and all its features described hereinabove are meant to encompass also the alginate that coats the SPIO NPs, namely the alginate that is used to prepare the alginate-coated SPIO-NPs. Hence, the alginate-coated SPIO-NPs are coated with alginate which exhibits a viscosity average molecular weight that ranges from 1 kDa to 1000 kDa, from 1 kDa to 500 kDa, from 1 kDa to 100 kDa, from 50 kDa to 200 kDa, or from 2 kDa to 50 kDa.

Alternatively, the alginate used for either SPIO-NPs coating or as free alginate exhibits a viscosity average molecular weight that ranges from 10-50 kDa, 20-50 kDa, 20-40 kDa, or have an average molecular weight of about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200 kDa and higher, up to 500 kDa or up to 1000 kDa, including any value therebetween.

It is known that gel fibers derived from low M/G ratio alginate is stronger than that from high M/G ratio alginate, and hence, that the M/G ratio of the alginate may govern at least some of the properties exhibited by alginate. Thus, is to be understood the term "alginate", referring to either SPIO-NPs coating or the free alginate, is meant to encompass alginates having M/G unit ratio that ranges from 0.1 to 10, from 0.2 to 5, or from 0.5 to 2, or have M/G ratio of about 0.1, 0.3, 0.6, 0.9, 1 or 2. Superparamagnetic iron oxide nanoparticles coated and stabilized by alginate (alginate-coated SPIO-NPs) can be prepared readily, as described, for example, by Ma et al. in Int. J. Pharm. 333, 177-186 (2007), which is incorporated by reference as if fully set forth herein.

Typical alginate-coated SPIO-NPs which are suitable in the context of the present invention, include, but are not limited to, Fe₃0₄ nanoparticles exhibiting a core diameter of 5-10 nm, which can give rise to alginate-coated SPIO-NPs exhibiting a hydrodynamic diameter of 100 nm to 600 nm.

It has been shown that the higher the molecular weight of the alginate, the more it is conformationally restricted to form alginate-coated SPIO-NPs with higher iron content. In addition, although G sequences of alginate are superior to complexes with iron ions compared to M sequences, complex formation is possible with both G and M sequences since the iron content is similarly produced with alginate having M/G ratio of 1.5 or alginate having M/G ratio of 0.6. In addition, the Fe₃0₄ content in alginate- coated SPIO-NPs produced with 1 percent by weight of an alginate having a viscosity average molecular weight of 100-200 kDa and an M/G ratio of 1.5 can reach up to 50 percent by weight iron oxide.

Typical magnetic measurements of such alginate-coated SPIO-NPs show superparamagnetism of anMs of 30 to 50 emu/g. In addition, alginate-coated SPIO-NPs exhibit a similar or lower Tj relaxivity while T₂ relaxivity is high, making alginate- coated SPIO-NPs useful as a negative contrast agent.

According to some embodiments of the present invention, the alginate-coated SPIO-NPs are dispersible or suspendable in aqueous media. Herein, the terms "dispersible" or "suspendable" are used to describe the capability of a single molecular entity to be dispersed or suspended in an aqueous solution or media. When used in the context of the nanoparticles described herein, these terms can be regarded as equivalent to "dissolvable" or "soluble" in aqueous solutions (e.g., water) as long as no precipitation and/or no formation of larger entities occur.

Alginate-coated SPIO-NPs are capable of forming aggregates in the presence of calcium ions. Hence, the methods presented herein make use of technologies such as magnetic resonance and dynamic light scattering (DLS) as well as other spectroscopic techniques, which can monitor formation, changes in formation and/or level of formation of these aggregates in a sample, as well as changes in suspendability thereof. The formation and/or level of formation of these aggregates can be correlated to the presence and/or level of calcium ions in the sample, as demonstrated in Example 1, Figures 2A-D, in the Examples section that follows below. As can be seen in the data shown hereinbelow, the aggregates may be dispersible or suspendable, and may also grow in size and precipitate. The precipitation of the aggregate can be detected by a clear signal inversion (see, Figures 2A-D), hence provide additional insight to the levels of calcium in the sample.

In the context of the present embodiments, the phrase "determining a presence and/or a level of calcium ions in aqueous media", as used herein, refers to the quantitative and/or qualitative correlation between the presence, number, size and dispersibility or suspendability of calcium-derived aggregates of superparamagnetic iron oxide nanoparticles (SPIO-NPs) coated with alginate. The correlation can be standardized against a set of standard solutions/samples wherein the calcium ions concentration is known.

The method presented herein, according to some embodiments of the invention, is suitable for determining the presence and/or the level of calcium ions in a sample, whereby the calcium ions concentrations can range widely from about 100 μΜ or lower to about 5 mM or higher. In some embodiments, the method presented herein, according to some embodiments of the invention, is suitable for determining the presence and/or the level of calcium ions in a sample, whereby the calcium ions concentrations range from about 100 μΜ or about 5 mM, or from about 250 μΜ to about 4 μΜ.

Different tissues contain calcium in different concentrations. For example, calcium (mostly Ca3(P0₄)2 and some CaS0₄) is the most prevalent element of bone and calcified cartilage. In mammalians about 99 percent of the total body content of calcium is present in the bones, which is largely unavailable for exchange (low bioavailability). Within a typical living cell the intracellular concentration of calcium ions is about 100 nM, but it can increase by 10 to 100-folds during various cellular processes. The intracellular calcium level is kept relatively low with respect to the extracellular fluid, by an approximate magnitude of 12,000-fold. For example, the physiological concentration of Ca²⁺ in fetal bovine serum (FBS) is about 2.5 mM. This gradient is maintained through various ATP-driven plasma membrane calcium pumps, as well as a sizable storage within intracellular compartments.

As calcium concentration varies extensively across and within various biological samples, the method presented herein is capable of obtaining meaningful results across a wide range of physiologic calcium concentrations. As presented hereinabove, presently known methods using Ca²⁺ indicators for magnetic resonance techniques, such as those based on SPIO-NPs functionalized with the calcium-sensing protein calmodulin, were found to be limited to detection of Ca²⁺ at concentrations near 1 μΜ and below due to saturation of the system at higher calcium concentrations.

As presented hereinabove, the present inventors have uncovered that formation of aggregates of alginate-coated SPIO-NPs in the presence of calcium ions, and in correlation to calcium ions concentration, can be even more effectively performed when free alginate is added to the alginate-coated SPIO-NPs.

As further presented herein, the addition of free alginate may widen the range of detectable calcium concentration. Thus, the method presented herein further includes, according to some embodiments thereof, adding free alginate to the formulation of the alginate-coated SPIO-NPs. The free alginate can be added separately to the interrogated sample, or, in case of in vivo applications, can be co-administered to a subject along with the alginate-coated SPIO-NPs described herein.

Alginates have been described in details hereinabove. The types of alginates used as a free alginate can be similar or different than the alginate used for preparing the alginate-coated SPIO-NPs.

The phrase "pharmaceutically acceptable salt" refers to an ionized form of alginate species, which is suitable for the method presented herein in terms of the bio- kinetics and bio-availability characteristics of the alginate-coated SPIO-NPs or free alginate species, and which does not evoke significant irritation when administered to a subject.

Figure 1 illustrates the effect of adding free alginate to a system having alginate- coated SPIO-NPs and calcium ions. Without being bound by any particular theory, it is assumed that the free alginate and the calcium ions bridge between alginate-coated SPIO-NPs to form larger aggregates, which grow larger with the growing concentration of free alginate and the calcium ions. Hence, without being bound by any particular theory, it is noted that the addition of free alginate increases the range of measurable calcium concentration using the method presented herein, by harnessing the tendency of free alginate and calcium ions to bridge (crosslink) between small aggregates of alginate-coated SPIO-NPs, and thereby form larger aggregates and/or super-aggregates.

The addition of free alginate can therefore be regarded as a mean to expand the range of calcium concentrations that can be determined using the methods presented herein and/or as a mean to increase the sensitivity of detection within a given calcium concentrations range. It is to be understood that the addition of free alginate is optional and that the method can be used to detect and determine calcium in a sample without the addition of free alginate.

The phrases "relatively high concentrations of calcium (Ca² )" or "high calcium levels", as used herein, refer to calcium concentrations higher than 0.2 mM, higher than 1 mM, higher than 2 mM or higher than 5 mM of Ca²⁺ ions.

Further without being bound by any particular theory, it is noted herein that free alginate can be used to control precipitation of large alginate-coated SPIO-NP aggregates in high concentrations of calcium ions, as demonstrated in the Examples section that follows below.

The present inventors have shown that the ratio between free alginate and alginate-coated SPIO-NPs, as well as the molecular weight of the added free alginate, can be used to determine at which Ca²⁺ concentration aggregation will occur, thus enabling to fine tune the signals which are detected in MR imaging, and thereby determine the Ca²⁺ levels in a sample, according to embodiments of the present invention.

The present inventors have also shown that the amount of free alginate added to the sample also increases the selectivity of alginate-coated SPIO-NPs towards Ca²⁺ versus Mg²⁺, which may generate false results against calcium, as discussed herein. As demonstrated in the Examples section that follows, addition of free alginate increased the imaging selectivity towards calcium, and the alginate-coated SPIO-NPs were capable of being used to detect Ca²⁺ and perform MR imaging at different Ca²⁺ concentration also in the presence of 0.5 mM Mg²⁺, which is typical for physiological samples. The amount of the optional free alginate additive is correlated to the sub-range of high calcium levels. Thus, for the sub-range of high calcium levels, from 2 mM to 5 mM, the method may be effected by the addition of free alginate at a concentration that ranges from 0.02 to 0.06 percent by weight of the total weight of the sample.

Accordingly, for the sub-range of high calcium levels, from 0.2 mM to 2 mM, the method is effected by the addition of free alginate at a concentration that ranges from 0.002 to 0.02 percents by weight of the total weight of the sample.

According to some embodiments of the present invention, the method presented herein is suitable for continuous monitoring ("real-time" monitoring) as well as interval- based monitoring of changes in the calcium levels in a sample wherein the calcium levels may vary and change due to processes taking place in the sample (e.g., a plant or an animal). The method presented herein is particularly suitable for monitoring fluctuations in calcium levels in a continuous or interval time regimes in a live as well as in an inanimate sample since it is based on non-invasive, non-destructive and non- mutilating spectroscopic technology (MR) wherein data can be collected from an intact sample in very short measuring time.

Such changes in calcium levels may occur due to change in biological activity of a living sample and/or as a response of the sample to manipulation of calcium levels effected intentionally. One exemplary case of medicinal interest wherein the method presented herein can be of use, is the change in calcium levels which are due to a change in brain activity and vice versa, since change in calcium level affect calcium channels and thus affects brain activity.

According to some embodiments, the method presented herein is effective for in vitro calcium determination by MRI in physiological media such as cell cultures, samples of bodily, samples of bodily tissues or organs, and for in vivo calcium determination by MRI, by imaging a bodily site of animals or human subjects.

The phrase "physiological medium", as used herein, refers to the combined substances (mostly solutes) and solvents (mostly water) which constitute the medium which can support, at least partially, the physical and biochemical functions of living organisms. In the context of the present embodiments, physiological medium may be a saline solution, other living cells-supporting solutions, bodily fluids, serum, blood, urine, bodily sites, cell cultures, and in its wide scope, any tissue and organ. The term "tissue" as used herein, refers to an aggregate of cells of an organism that have similar structure and operate in unison to carry out a specific set of functions form an organ. Non-limiting examples of tissues include epithelial, nerve, connective, muscle and vascular animal tissues, or meristematic (apical meristem and cambium), protective (epidermis and cork), fundamental (parenchyma, collenchyma and sclerenchyma) and vascular (xylem and phloem) tissues in plants.

The term "organ", as used herein, refers to a group of tissues that perform a specific function or group of functions. Non-limiting examples of animal organs include heart, lungs, brain, eye, stomach, spleen, muscles, bones, pancreas, kidneys, liver, intestines, skin, urinary bladder and sex organs, and in plants include roots, stems, leaves, flowers, seeds and fruits.

One of the problems facing mineral detection and determination in biologic samples is the similarities in the properties of some elements and their ions, such as divalent ions. The presently known methodologies for monitoring any given ion or mineral in a physiologic environments and samples, often suffer from large errors and skewed results due to cross-effect of the similar ions or minerals. Thus, the detection of calcium ions may be impaired by the presence of other metal ions, particularly of divalent metal ions such as magnesium ions, which are ubiquitous in physiological media. Thus, is some embodiments, the methodology presented herein can be efficiently performed in the presence of cations other than calcium ions, and more specifically in the presence of magnesium ions, since alginate exhibits lower affinity to cations of metals such as magnesium, as compared to its affinity to calcium ions, and therefore the signal detected for calcium-derived aggregation overshadows a signal that may arise from non-calcium-derived aggregation. The ability to provide quality data in the presence of magnesium ions, as found under typical physiologic conditions, provides a solution to the aforementioned problem of determining a presence and/or a level of calcium ions in a sample.

A capacity of determining a presence and/or a level of calcium ions in a sample, using the herein described methodology, in the presence of magnesium, is demonstrated in Example 1, Figure 5 in the Examples section that follows.

According to another aspect of some embodiments of the present invention, there is provided a composition, identified for use in determining a presence and/or a level of calcium ions in a sample by magnetic resonance spectroscopy (e.g., imaging), which includes a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate. The composition presented herein may be identified for use in combination with free alginate, as presented hereinabove. The free alginate can form a part of the composition as a single formulation, or can be utilized as an additive that is provided in a separate formulation.

According to another aspect of some embodiments of the present invention there is provided a use of a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate as a diagnostic agent for determining a presence and/or a level of calcium ions in a sample by magnetic resonance spectroscopy (e.g., imaging).

According to another aspect of some embodiments of the present invention there is provided a use of a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate in the manufacture of a diagnostic agent for use in a method of determining a presence and/or a level of calcium ions in a sample, biological tissues, organs, living animals or human subjects by magnetic resonance spectroscopy (e.g., imaging).

Further accordingly, there is provided a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate for use in determining a presence and/or a level of calcium ions in a sample, as defined herein, by magnetic resonance spectroscopy (e.g., imaging).

As discussed hereinabove, determining a presence and/or a level of calcium ions in a sample, be it in vitro or in vivo, is beneficial for monitoring medical conditions associated with calcium ions in an animal or human subject. Such a monitoring can be performed in vitro, by, for example, imaging samples of bodily fluids, tissues or organs taken from the subject, or, in vivo, by, for example, imaging bodily sites of the subject upon administering to the subject the superparamagnetic iron oxide nanoparticles coated with an alginate as described herein.

According to another aspect of the present invention, there is provided a method of monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject, namely a living human, a living animal or a sustained organ. The method is effected by: administering to the subject a diagnostically effective amount of a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate;

subjecting the subject to MR imaging; and

detecting a formation of aggregates of at least a portion of the plurality of superparamagnetic iron oxide nanoparticles coated with an alginate, wherein a formation and/or level of formation of said aggregates is indicative of the presence and/or level of calcium ions in an organ or tissue of the subject, thereby monitoring the presence and/or progress of the medical condition.

The phrase "diagnostically effective amount" in the context of administering to a subject a composition comprising alginate-coated superparamagnetic iron oxide nanoparticles, refers to the amount of nanoparticles which is sufficient to produce a detectable signal relevant to MRI.

Typical for the use of SPIO-NPs, as well as alginate-coated SPIO-NPs, the diagnostic effective amount refers to the concentration of iron (Fe). In some embodiments, a concentration of the alginate-coated SPIO-NPs in the aqueous solution is such that the iron concentration ranges from 0.01 mM to 1 mM.

Hence, the amount of alginate-coated SPIO-NPs in the composition presented herein, can be determined experimentally, and can be defined by the strength of the signal which is monitored by the MR detector at a specific bodily site (or another sample).

The composition containing the alginate-coated SPIO-NPs, with or without free alginate as described herein, can be administered as a single dose at the beginning of the imaging routine. Alternatively, the amount of the composition can be varied during the routine so as to achieve contrasting effects and in order to monitor changing calcium ion levels during the course of a process. Alternatively, the composition containing the alginate-coated SPIO-NPs can be administered at a constant rate (drip) while the MRI scan is performed at time intervals or continuously.

The composition containing the alginate-coated SPIO-NPs, with or without free alginate, can be administered to a live subject according to the bodily site which is being imaged. For example, the composition can be administered orally, rectally, intravenously, intraventricularly, intraperitoneally, intestinally, parenterally, intraocularly, intradermally, transdermally, subcutaneously, intramuscularly, transmucosally and/or by intrathecal catheter. Optionally, the composition containing the alginate-coated SPIO-NPs, with or without free alginate, can be administered by means of a medical device such as a catheter or a gastroscope that is designed for directly delivering the composition to the bodily site.

A typical MRI procedure can be performed using any NMR of MRI system using a multitude of single or multi-slices pulse sequences, or using 3D imaging methods as these terms are known in the art. MR images can be acquired immediately after or continuously after any medical conditions or functional stimulations, such that calcium levels require monitoring. Measurements can be perfumed on a single and/or multiple time points. Any numeric processing method can be used correlate the signals detected from the sample and quantify the effect of the aggregation of alginate-coated SPIO-NPs in the MRI images to calcium level.

According to another aspect of the present invention there is provided a use of a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate in the manufacture of a diagnostic agent for monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject by magnetic resonance imaging or spectroscopy (MRI or MRS).

According to another aspect of the present invention there is provided a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate, for use in monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject by magnetic resonance imaging or spectroscopy

(MRI or MRS).

In any of the methods and uses described herein, the superparamagnetic iron oxide nanoparticles coated with an alginate can be used in combination with free alginate, as described herein. The free alginate can be co-formulated with the superparamagnetic iron oxide nanoparticles coated with an alginate, or can be coadministered prior to, concomitant with, or subsequent to administering the superparamagnetic iron oxide nanoparticles coated with an alginate.

Medical conditions associated with physiologic calcium levels include, without limitations, hypocalcaemia, hypercalcaemia, constipation, eating disorders, chronic renal failure, severe acute hyperphosphatemia, ineffective PTH syndroms, pancreatitis, alkalosis, bone pain, kidney stones, psychotic behavior, depression, confusion, psychiatric overtones, cerebral ischemia, cardiac ischemia, cardiac arrest, heart failure, different types of cancer, neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson, multiple sclerosis, Alzheimer disease and more.

According to some embodiments, imaging is conducted on bodily sites in which calcium concentration is affected as a result of a medical condition (e.g., disease or disorder).

Exemplary bodily sites include, for example, a bone tissue, brain, heart, kidneys, spleen, a muscle tissue, and the like.

Without being bound by any particular theory, it is assumed that the changes in calcium levels in various bodily sites are monitored and controlled by brain activity, while the brain itself is one of these bodily sites wherein calcium ions play a key role in its activity. Hence, it is clear that the method presented herein can be used to image many biological processes and be used to diagnose many medical conditions, by utilizing the claimed methodology for imaging at least a portion of the brain of a subject.

According to some embodiments, each of the methods and compositions for monitoring a change in a level of calcium ions in a subject described herein, are for coadministering to the subject a diagnostically effective amount of alginate-coated superparamagnetic iron oxide nanoparticles, and a diagnostically effective amount of free alginate.

The phrase "diagnostically effective amount" in the context of co-administering free alginate with a plurality of alginate-coated SPIO-NPs, refers to the amount of free alginate with alginate-coated SPIO-NPs which is sufficient to produce a detectable signal (contrast) relevant to MRI, in certain conditions, such as for example, a range of relatively high concentrations of calcium and/or the presence of a high concentration of another metal ion.

Since MRI is substantially a technique for monitoring magnetic resonance differences in water molecules found in the sample, the contrasting effect (signal) produced by the alginate-coated SPIO-NPs, which is sensitive to the level of calcium ion, can be increased or decreased depending on the diagnostic needs. Hence, one way to determine the amount of alginate-coated SPIO-NPs to be administered and whether free alginate is required can be effected experimentally by performing a base experiment with a typical amount of alginate-coated SPIO-NPs per sample size (weight), followed by the addition of alginate-coated SPIO-NPs and/or free alginate so as to achieve fine adjustments of the signal.

As presented hereinabove, free alginate is added in order to allow the detection of relatively high levels of calcium, and the amount (concentration) of free alginate which is administered to the subject can be selected suitable for imaging a certain level of calcium, as presented in details hereinabove. Free alginate may be co-administered to the subject in the same formulation containing the alginate-coated superparamagnetic iron oxide nanoparticles, or in a separate formulation.

The stage of administration (e.g., prior to, concomitant with or subsequent to, and/or the concentration of the free alginate are determined as required per the guidelines provided hereinabove.

The phrase "diagnostic monitoring of a physiological process and/or a medical condition", as used herein, encompasses determination of a presence of the medical condition (e.g., as a part of routine check-up of a healthy subject or for diagnosing a subject suspected as having a medical condition); monitoring the progression of a medical condition or of a symptom thereof in a subject afflicted with the condition; and monitoring the responsiveness to a therapy of a medical condition (thus forming a part of the treatment of subject afflicted with the condition).

It is to be understood that the methods and compositions presented herein can be used for general and comparative studies of any physiological process for any scientific purpose regardless of an association to a medical condition.

In any of the methods and uses described herein, the alginate-coated SPIO-NPs can be utilized either per se, or as a part of a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.

The phrase "pharmaceutical composition" encompasses use of the composition for diagnostic uses, and can therefore be referred a diagnostic composition in the context of some of the present embodiments.

In some embodiments, the pharmaceutical composition is identified for use in determining a level and/or presence of calcium ions. In some embodiments, the pharmaceutical composition is identified for use in diagnostic monitoring of a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject by magnetic resonance imaging or spectroscopy (MRI or MRS). In some embodiments, the pharmaceutical composition is packaged in a packaging material and is identified in print, in or on the packaging material, for use in the indicated diagnostic method, as described herein.

As used herein the phrase "pharmaceutical composition" refer to a preparation of alginate-coated SPIO-NPs with or without free alginate as described herein, with other chemical components such as pharmaceutically acceptable and suitable carriers and excipients, and optionally with additional active agents, such as another contrasting agent. The purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject.

Hereinafter, the term "pharmaceutically/diagnostically acceptable carrier" refers to a carrier or a diluent that does not cause significant irritation to an organism and does not inhibit the distribution, magnetic properties or otherwise activity and properties of the administered compound.

Formulations for parenteral administration may include, but are not limited to, sterile solutions which may also contain buffers, diluents and other suitable additives. Pharmaceutical/diagnostic compositions for use in accordance with embodiments of the invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the composition into preparations which can be used diagnostically. Proper formulation is dependent upon the route of administration chosen. Toxicity and diagnostic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in experimental animals, e.g., by determining the EC50, the IC50 and the LD50 (lethal dose causing death in 50 % of the tested animals) for a subject combination of alginate-coated SPIO NPs and/or free alginate. The data obtained from these assays and animal studies can be used in formulating a range of dosage for use in human based on relative body mass and distribution in a particular bodily site.

The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the diagnostic practitioner in view of the diagnostic conditions. In general, the dosage is related to the efficacy of the active ingredient which, in the context of embodiments of the invention, is related to the diagnostic capacity of the alginate-coated SPIO NPs and/or free alginate, and the particular pharmacokinetics and pharmacology thereof for absorption, distribution, metabolism, excretion and toxicity (ADME-Tox) parameters. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of a present affliction, the manner of administration, the judgment of the diagnostic practitioner, the imaging technician, and/or the prescribing physician.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA (the U.S. Food and Drug Administration) approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as, but not limited to a blister pack or a pressurized container (for inhalation). The pack or dispenser device may be accompanied by instructions for use. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising alginate-coated SPIO NPs, either alone or in combination with free alginate as described herein and/or another diagnostic probe or agent, formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for diagnosis of an indicated condition, as is detailed herein.

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

The word "exemplary" is used herein to mean "serving as an example, instance or illustration". Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

The word "optionally" is used herein to mean "is provided in some embodiments and not provided in other embodiments". Any particular embodiment of the invention may include a plurality of "optional" features unless such features conflict.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Materials and Experimental Methods

Preparation of alginate-coated SPIO nanoparticles:

Alginate-coated SPIO nanoparticles were prepared according to the procedure for producing magnetite nanoparticles via the chemical co-precipitation technique. Briefly, ferrous sulfate (FeS0₄-7H₂0, 208 mg) and ferric chloride (FeCl₃-6H₂0, 280 mg) were dissolved in 10 ml of distilled water under argon atmosphere. The solution was warmed to 60 °C and 5 ml of 5 M sodium hydroxide (NaOH pellets) solution was added dropwise to the stirred iron salts solution. 100 μΐ of 1 % (w/w) 30 kDa alginate (PRONOVA UP VLVG by NovaMatrix, FMC Biopolymers, Drammen, Norway) solution was added. The mixture was stirred for additional 30 minutes at 60 °C and then at 80 °C for 1 hour. The brown suspension was sonicated for 20 minutes and centrifuged for 20 minutes at 8500 rpm. The water poured and 15 ml of distilled water were added to the precipitate. The sonication and centrifugation processes were repeated twice to obtain transparent brown solution of alginate-coated SPIO-NPs in water. Dynamic Light Scattering (DLS):

The averaged particles sizes were determined by DLS experiments (using ALV- Particle Sizer), which were performed in 800 μΐ solutions (10 mM HEPES buffer, pH=7.2) composed of 400 μΐ alginate-coated SPIO-NPs solution and 400 μΐ of the tested solution (H₂0 for reference or Ca²⁺ 0.5 mM or Mg²⁺ 0.5mM). The tested solutions were incubated for three hours at 37 °C before DLS measurements.

MRI:

10-12 capillaries with the tested solutions were well oriented and placed in 8 mm stand, which was inserted into 10 mm NMR tube containing Fluorinert™ (by 3M™). Each capillary contained alginate-coated SPIO-NPs solution, 10 mM HEPES buffer (pH of 7.2) and the tested solution in a way that the final concentration of Ca²⁺ or Mg²⁺ can be determined.

MPJ experiments were performed using a 8.4T spectrometer (Bruker, Germany).

T₂ weighted images were acquired using the spin echo sequence with 16 echoes

(TR=5000 ms and TE = 10, 20, 160 ms). Four mm slice was acquired with a field of view (FOV) of 1.28 x 1.28 cm² and 256 ^x 128 digital resolution reconstructed to a 256 x 256 matrix. Total acquisition time was about 42 minutes and T₂ weighted images were acquired up to 5 hours after preparation. Percentage changes in the MRI signal intensity was calculated as presented in Equation 1 below:

Equation 1

Averaged Signal Intensity (smaple) - Averaged Signal Intensity (reference)

Averaged Signal Intensity (reference) MRI imaging of the brain in vivo was performed on a 7T/30 cm Biospec MRI scanner (Bruker, Germany). T₂ ^* and ₂-weighted MR images were collected. T₂- weighted MR images were acquired using the spin echo sequence (TR/TE=3500/40, 60, 80 ms) and ₂ ^*-weighted images were collected by the gradient echo sequence (TR/TE=268/6, 10, 15ms). In these MR images one mm slices were acquired with a FOV of 2 x 2 cm² and 256 x 128 digital resolution reconstructed to a 256 x 256 matrix. MRI was performed under isofluorane. The total collection time of the MRI protocols was about 30 minutes. EXAMPLE 1

The detection of Ca²⁺ ions using alginate-coated SPIO-NPs was tested in three different techniques: visually, using dynamic light scattering (DLS), and by MRI, as shown in Figures 2A-D.

Figures 2A-D present various modes of expressing the effect of Ca²⁺ on alginate-coated SPIO-NPs, wherein the visual inspection of the alginate-coated SPIO- NPs 3 hours after the addition of H₂0, 0.5 mM/1.0 mM of Ca²⁺ or Mg²⁺, reveals clear precipitation only in the 1 mM Ca²⁺ solution (Figure 2A), the particle's size analyses of alginate-coated SPIO-NPs 3 hours after the addition of H₂0, 0.5 mM Ca²⁺ or 0.5 mM Mg²⁺ as obtained from DLS measurements, reveal a clear increase in the size of the alginate-coated SPIO-NPs in the presence of 0.5 mM Ca²⁺ (Figure 2B), ₂-weighted MR images (TR TE=5000/20 ms) of aqueous solutions of alginate-coated SPIO-NPs with 0.002 % of free alginate in the absence and in the presence of 0.5 mM/1.0 mM of Ca2+ or Mg²⁺, show that Ca²⁺ has dramatic effect on the contrast, while Mg²⁺ has practically no effect on the signal intensity (Figure 2C), and in the bar plot of percentage change in the MR signal intensity, of the ^-weighted MR images shown in Figure 2C, as a function of the reference sample (Figure 2D).

As can be seen in Figure 2 A, brown sediments were observed in the 1.0 mM Ca²⁺ solution where alginate-coated SPIO-NPs aggregate and precipitate.

As can be seen in Figure 2B, the aggregation of alginate-coated SPIO-NPs indeed occur at lower levels of Ca²⁺ after 3 hours from the addition of the alginate- coated SPIO-NPs to the 0.5 mM M²⁺ solution. The particles size detected by DLS was significantly larger in the Ca²⁺ solution (150 ± 10 nm) compared with the H₂0 reference (70 ± 10 nm) or Mg²⁺ (80 ± 15 nm) solutions. These differences in the measured dynamic radii demonstrate that 0.5 mM Ca²⁺ results in dispersible or suspendable alginate-coated SPIO-NPs aggregates. However, in the presence of Mg²⁺, the observed dynamic radii were very similar to that obtained in the presence of water (reference sample) implying that no significant particles aggregation occurs under these conditions.

These results are supported by MR images presented in Figure 2C, which show that the MRI contrast is influenced by the Ca²⁺ level. As can be seen in Figure 2C, at Ca²⁺ concentration of 500 μΜ (0.5 mM) the MR image is darker in spin echo images (TPv/TE = 5000/20 ms) compared to the alginate-coated SPIO-NPs water reference tube, while at higher levels of Ca²⁺, i.e., 1.0 mM, the MR image is brighter compared to the alginate-coated SPIO-NPs solutions in the absence of any salt or even in the presence of Mg²⁺. The percentage differences in the MR signal intensity compared to the reference alginate-coated SPIO-NPs solution without M²⁺ ions are given in Figure 2D, and show the sensitivity and the selectivity of alginate-coated SPIO-NPs to Ca²⁺ MR imaging.

The results presented in Figures 2A-D indicate that the aggregate sizes of our alginate-coated SPIO-NPs as well as their aggregation level depend on Ca²⁺ concentration. At lower Ca²⁺ levels the aggregates are small enough to remain dispersible or suspendable and therefore the ₂-weighted MR image is darker compared to non-aggregated particles (present in the presence of water or Mg²⁺). At Ca²⁺ of 1.0 mM the obtained aggregates are presumably bigger and non-dispersible/suspendable (Figure 2A) and therefore the ^-weighted MR image is brighter as a result of the removal of the alginate-coated SPIO-NPs from the detected solution.

Interestingly, the selectivity of the alginate-coated SPIO-NPs to Ca²⁺ as compared to its main interfering cation the divalent cation Mg²⁺ was observed only in the presence of traces of free alginate. In addition, it was found that the amount and the characteristics of the free alginate added may control not only the specificity of the calcium MRI biomarker according to the present embodiments, but also allow the detection of different ranges of concentrations of Ca²⁺, as shown in Figure 3.

Figure 3 presents ₂-weighted MR images (TR/TE=5000/ 10ms) of aqueous solution containing alginate-coated SPIO-NPs and the indicated Ca²⁺ concentrations (in mM), acquired 4 hours after the addition of Ca²⁺ to different alginate-coated SPIO-NPs solutions which are placed in the matrix by rows, wherein row "a" is a solution without free alginate, row "b" is a solution with 0.002 % of 14 kDa alginate, row "c" is a solution with 0.002 % 30 kDa alginate, row "d" is a solution with 0.02 % of 14 kDa alginate, and row "e" is a solution with 0.02 % 30 kDa alginate. These ₂-weighted MR images show that with the addition of different amount of free alginate one can tune the sensitivity of the alginate-coated SPIO-NPs to different ranges of Ca²⁺ concentration.

As can be seen in Figure 3, the alginate-coated SPIO-NPs form dispersible or suspendable aggregates at low Ca²⁺ concentrations (250 μΜ) without the addition of free alginate, resulting in a darker ^-weighted image (row "a" in Figure 3). In this case, however, at Ca²⁺ levels greater than 500 μΜ, non-dispersible/suspendable NP aggregates are formed, which are expelled from the aqueous solution, leading to an intense MR signal, compared to the reference sample. Rows "b-e" in Figure 3 show the dependency of the obtained MRI contrast, in different Ca²⁺ concentrations, on the amount and molecular weight of free alginate added. The results suggest that the ratio between free alginate and alginate-coated SPIO-NPs as well as the molecular weight of the added alginate determine at which Ca²⁺ concentration aggregation will occur thus enabling to fine tune the platform for Ca²⁺ MR imaging in aqueous solutions at different concentration ranges of Ca²⁺.

It is noted that the amount of free alginate added to the aqueous solution of alginate-coated SPIO-NPs also increases the selectivity of alginate-coated SPIO-NPs towards Ca²⁺ as compared to Mg²⁺, which is the most interfering cation when Ca²⁺ detection is required, as shown in Figure 4.

Figures 4A-E present comparative plots of the percentage of signal intensity change in the ^-weighted MR images (TR/TE=5000/20 ms) 3-5 hours after the addition of the indicated concentrations of Ca²⁺ and Mg²⁺ ions to alginate-coated SPIO- NPs solutions, in the presence of different amount of 30 kDa and 14 kDa alginates, wherein no addition of free alginate is shown in Figure 4A, addition of 0.002 % of 30 kDa alginate is shown in Figure 4B, with 0.02 % 30 kDa alginate is shown in Figure 4C, with 0.002 % of 14 kDa alginate is shown in Figure 4D, with 0.02 % 14 kDa alginate is shown in Figure 4E.

As can clearly be seen in Figures 4A-E, addition of free alginate increases the selectivity towards calcium, and that the alginate-coated SPIO-NPs and the above formulation are capable of detecting Ca²⁺ and imaging different Ca²⁺ concentration also in the presence of Mg²⁺, at physiological levels, namely, 0.5 mM.

Figures 5A-C present ₂-weighted MR images (TR/TE=5000/20ms) accompanied with a plot of the percentage change in the MR signal intensity (relative to a sample with no calcium) of the water in aqueous solution of the alginate-coated SPIO- NPs in the presence of 0.5 mM Mg²⁺ after the addition of 0.002 % 30 kDa alginate (Figure 5A), after addition of 0.02 % 30 kDa alginate (Figure 5B), and after addition of 0.02 % 30 kDa alginate 0.1 mM changes in Ca²⁺ levels (Figure 5C). As can be seen in Figures 5A-C, the MR images as well as the graphs demonstrate that the methodology described herein can be used to detect Ca²⁺ in different ranges of concentrations even in the presence of magnesium. For example, in the presence of 0.002 % of free alginate solution the degree of aggregation of the alginate-coated SPIO-NPs is moderate at 0.5 mM Ca²⁺ resulting in dispersible or suspendable aggregates which reduce the signal intensity by 53 % as compared to the reference solution (Figure 5A). At higher Ca²⁺ levels, e.g., higher than 0.75 mM, large non-dispersible/suspendable aggregates are formed, resulting in brighter MRI images with a much higher signal intensity of more than 200 % as compared to the reference solution. Interestingly, as shown for the Mg²⁺-free solutions (Figure 3), larger amount of free alginate in the detecting media (0.02 %), allow MR detection of higher calcium levels. Here, some decrease in the signal intensity was observed for Ca²⁺ concentrations of 0.5, 0.75 and 1.0 mM. High MRI signal was observed only for 1.5 mM Ca²⁺, where massive, non-dispersible/suspendable aggregates were obtained.

Examination of Ca²⁺ concentrations between 0.8 mM and 1.3 mM shows that alginate-coated SPIO-NPs are sensitive to 100 μΜ changes in Ca²⁺ levels (Figure 5C). Such sensitivity may be relevant to image pathologies, as well as the function of normal and diseased brain, where small changes in extracellular calcium are obtained [Kristian et al. Exp. Brain Res. 120, 503-509 (1998), or to detect Ca²⁺ variations during intense neuronal stimulation [Breitwieser, G. E. Int. J. Biochem. Cell Biol. 40, 1467-1480 (2008)].

Figures 6A-B present ₂-weighted MR images (TR/TE=5000/ 10ms (Figure 6 A) and 5000/20 ms (Figure 6B)), 4 hours after the addition of different concentration of Ca²⁺/Mg²⁺ (0-4.0 mM) to aqueous solution of alginate-coated SPIO-NPs to which 0.05 % of 30 kDa alginate was added, showing the ability to detect very high concentration of calcium, showing that despite the pronounced changes found upon addition of Ca²⁺, no change in signal intensity was observed in the ^-whighted MR images upon addition of Mg²⁺.

As can be seen in Figures 6A-B, high level of aggregation (bright MR images) was observed under these conditions only at Ca²⁺ concentrations greater than 2.0 mM. It is noted herein that such aggregation was not observed in the presence of equal, high Mg²⁺ levels. These findings imply that aggregation of the alginate-coated SPIO-NPs occurs primarily in the presence of Ca²⁺ and not in the presence of Mg²⁺.

As can further be seen in Figures 6A-B, increasing the free alginate content in the detecting solution (in the presence of 0.5 mM Mg²⁺), allows one to fit this flexible Ca²⁺ MR imaging platform to detect extremely high calcium levels (> 2.0 mM) at pH of 7.2 and 37 °C.

EXAMPLE 2

The successful use of alginate-coated SPIO-NPs as specific probe for Ca²⁺ MR imaging at variable levels of Ca²⁺ in aqueous solutions was further tested in different physiological media in order to investigate the probe in more complex solutions that simulate biological conditions.

Fetal bovine serum (FBS) was first used as a medium. Thus, Ca²⁺ solutions at variable concentrations were added to FBS samples (containing 10 mM HEPES buffer, pH=7.2) which contain physiological concentration of Ca²⁺, namely 2.4-2.6 mM, [Kim et al. Angew. Chem. Int. Ed. 48, 4138-4141 (2009)].

Figures 7A-B present comparative bar-plots of the percentage change in the signal intensity in ^-weighted MR images (TR/TE=3000/20ms), 45 minutes after addition of different concentration of Ca²⁺ to fetal bovine serum (FBS) solutions containing alginate-coated SPIO-NPs (Figure 7A) and to Dulbecco's Modified Eagle Medium (DMEM) solutions containing alginate-coated SPIO-NPs (Figure 7B), wherein all samples had 0.02 % of 30 kDa alginate added thereto, showing that the sensitivity to different concentration of calcium exists even in biological media.

As can be seen in Figure 7A, different Ca²⁺ concentrations from 0.5 to 2.0 mM can be detected using our SPIO-alginate particles using ₂-weighted MR images.

As can be seen in Figure 7B, similar MR response was observed also for different calcium levels in DMEM, which is a widely used medium for culture cells.

It is noted herein that the examined physiological media contained physiological levels of Ca²⁺ and other ions and still our alginate-coated SPIO-NPs were able to detect the addition of Ca²⁺ to these media. For change of less than 1.5 mM of Ca²⁺, only water-dispersible/suspendable aggregate are formed within the first 45 minutes and a reduction in MR signal intensity is observed in the ^-weighted image. For addition of 2 mM and more of Ca²⁺ an increase in the signal intensity was observed because of the formation of large non-dispersible/suspendable SPIO-alginate NP aggregates, as seen in Figure 7.

EXAMPLE 3

In order to corroborate the proposed mode of action, according to which alginate-coated SPIO-NPs aggregate in the presence of Ca²⁺ as a result of the alginate coating, the MRI experiments were performed in the presence of Feridex®, by Advanced Magnetic Industries Inc. Feridex® is a dextrane coated superparamagnetic iron oxide nanoparticles that also act as "negative" contrast agent. No Ca²⁺ sensitivity as well as Ca²⁺ selectivity (over Mg²⁺) were observed for the dextrane coated SPIO- NPs.

EXAMPLE 4

To challenge further the ability of alginate-coated SPIO-NPs to detect calcium in biological relevant setting, alginate-coated SPIO-NPs were added to solutions obtained from normal and ischemic cell culture in DMEM (Dulbecco/Vogt modified Eagle's minimal essential medium). After the cells were incubated in those solutions for 4 hours, the cells were removed by centrifugation and the alginate-coated SPIO-NPs containing 0.002 % or 0.02 % 30 kDa alginate were added and monitored by Τχ- weighted MRI.

These images were compared to images of the reference solutions which are alginate-coated SPIO-NPs containing 0.002 % or 0.02 % 30 kDa alginate added to the same media which had no contact with cells.

Figure 8 presents a bar-plot of the percentage change in the signal intensity in 2-weighted MR images (TR/TE=3000/40 ms) of solutions with and without cells, 30 minutes after addition of alginate-coated SPIO-NPs with 0.002 % (bars "a" and "b") or 0.02 % (bars "c" and "d") of 30 kDa alginate to DMEM (bars "a" and "c") or ischemic DMEM (bars "b" and "d"), showing that in ischemic DMEM there is a release of calcium which can be detected by our MRI probe, and showing that the sensitivity to different concentration of calcium exists even in biological media (data obtained from 11 different experiments, n=l 1). As can be seen in Figure 8, a significant change in the signal was detected only in the solution obtained from the ischemic cell culture, where a higher Ca²⁺ concentration is most likely to be found. As can be seen in Figure 8, only in the ischemic DMEM the reduction of the signal intensity was observed, suggesting the formation of dispersible or suspendable aggregates of alginate-coated SPIO-NPs. It should be noted that the presented results are averages of four different experiments.

EXAMPLE 5

The toxicity of the alginate-coated SPIO-NPs was evaluated for their safety in medical applications. This was achieved by monitoring the survival rate of C8-D1A astrocyte cells (obtained from ATCC) in the absence and presence of alginate-coated SPIO-NPs for 24 hours.

Figure 9 presents a comparative bar-plot of the percentage of cells survival at 4, 8 and 24 hours after exposure of C8-D1A astrocyte cells to HEPES buffer 10 mM as control and to SPIO-alginate NP diluted 1 : 1 with HEPES buffer 20 mM.

As can be seen in Figure 9, no toxic effect of the alginate-coated NPs was observed on C8-D1A astrocyte cells. The percentage of cells survival was 100 % after 4, 8 and 24 hours post exposure of the cells to alginate coated NPs and was very similar to the cell survival after exposed to HEPES buffer 10 mM, used as control for benign media.

EXAMPLE 6

The performance of the alginate-coated SPIO-NPs according to the present embodiments was challenged in vivo in the rat brain following quinolinic acid (QA) intoxication. QA is a known experimental model for Huntington disease (HD) which is accompanied by irreversible damage to the striatum.

QA 150 nmol in 1 μΐ of saline were injected to the right striatum while 1 μΐ of saline were injected into the left striatum. Seven days after the QA injection 2 μΐ solution of alginate-coated SPIO-NPs were injected to the two striatum areas (a subcortical part of the telencephalon, which is the anterior part of forebrain). At time- points of 1-2 hours, 3 days and 7 days following the injection, the rats brain were imaged in vivo using a 7T/30 cm Biospec MRI scanner. Both T₂ - (TR/TE=208/6, 10 ms) and r₂-weighted (TR/TE=3500/60, 80 ms) MR images were collected 1-2 hours, 3 days and 7 days post SPIO-NPs injection.

Figures 10A-B present two in vivo ₂-weighted MR images

(TR/TE=3500/60ms) taken from brains of two rats, seven days after the injection of quinolinic acid (150 nmol of quinolinic acid in 1 μΐ of PBS) to the right hemisphere, and 1 μΐ of PBS to the left hemisphere, and a few hours after the bilateral injection of 2 μΐ of the aqueous solution of (Figure 10A) alginate-coated SPIO-NPs, and the bilateral injection of FERIDEX® (Figure 10B).

As can be seen in Figures 10A-B, the darker area (hypointense) on the right lobe in Figure 10A, matches the quinolinic acid lesion which is shown only in the damaged striatum, in which Ca²⁺ concentration should be higher.

It was found that 1-2 hours and 3 days following alginate-coated SPIO-NPs injection to striatum in both hemispheres, black areas were found in the images only in the striatum where the QA lesion was apparent, as expected, only for the group treated with alginate-coated SPIO-NPs. The QA caused cells death in the striatum, which in turn resulted in Ca²⁺ accumulation in the damaged area. In that area much larger negative contrast was found. The black area in the images appeared only on one side of the brain (Figure9A), showing that our alginate-coated SPIO-NPs can indeed detect the increase in the calcium concentration also in brain in vivo. The ratio of hypointense digital image pixels between the right and left hemispheres, injected with QA or PBS respectively, as was extracted from 16 rats injected bilaterally with alginate-coated

MNPs and 8 rats injected bilaterally with FERIDEX®.

Figure 11 presents a comparative bar-plot of hypointense pixels counted in weighted digital images taken from 16 rats injected bilaterally with alginate-coated SPIO-NPs and 8 rats injected bilaterally with FERIDEX®, showing the ratio between the right and left hemispheres injected with QA and PBS respectively (p-values were <

0.05).

As can be seen in Figure 11 , both the ₂-weighted MR images and the graph presented in Figure 11 clearly show that the difference between the two hemispheres treated with FERIDEX® show little difference, while the difference observed in MR images of the group treated with our alginate-coated SPIO-NPs is highly notable. More dark pixels where observed in the QA intoxicated striatum but only for animals that were treated bilaterally with our alginate-coated SPIO-NPs.

Both the MR images presented in Figures 10A-B and the graph presented in Figure 11 demonstrate that the difference between the right and left hemispheres injected with QA or PBS respectively, is much more pronounced for the group treated with our alginate-coated SPIO-NPs in comparison to the group treated with FERIDEX®.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of determining a presence and/or a level of calcium ions in a sample, the method comprising:

subjecting the sample to magnetic resonance imaging or spectroscopy, to thereby detect a formation of aggregates of at least a portion of said superparamagnetic iron oxide nanoparticles coated with said alginate, wherein a formation and/or level of formation of said aggregates is indicative of the presence and/or level of calcium ions in the sample, thereby determining a presence and/or a level of calcium ions in the sample.

2. The method of claim 1, being for determining a presence and/or level of calcium ions in a range of from 100 μΜ to 5 mM.

3. The method of any of claims 1-2, further comprising contacting the sample with a free alginate.

4. The method of claim 3, wherein a concentration of said free alginate ranges from 0.02 to 0.06 percent by weight of the total weight of the sample.

5. The method of claim 4, being for determining a presence and/or level of calcium ions in a range of from 2 mM to 5 mM.

6. The method of claim 3, wherein a concentration of said free alginate ranges from 0.002 to 0.02 percent by weight of the total weight of the sample.

7. The method of claim 6, being for determining a presence and/or level of calcium ions in a range of from 0.2 mM to 2 mM.

8. The method of any of claims 1-7, being for determining a presence and/or a level of calcium ions in the presence of ions of a metal other than calcium.

9. A composition comprising a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate, the composition being identified for use in determining a presence and/or a level of calcium ions in an aqueous medium by magnetic resonance imaging or spectroscopy of a sample.

10. A plurality of superparamagnetic iron oxide nanoparticles coated with an alginate, for use in determining a presence and/or a level of calcium ions in an aqueous medium by magnetic resonance imaging or spectroscopy of a sample.

11. Use of a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate in the manufacture of a diagnostic agent for determining a presence and/or a level of calcium ions in a sample by magnetic resonance imaging or spectroscopy.

12. The composition of claim 9, the plurality of superparamagnetic iron oxide nanoparticles coated with an alginate of claim 10 or the use of claim 11, being for determining a presence and/or level of calcium ions having a concentration at a range from 100 μΜ to 5 mM.

13. The composition of claim 9, further comprising a free alginate.

14. The plurality of superparamagnetic iron oxide nanoparticles coated with an alginate of claim 10 or the use of claim 11 , wherein said plurality of superparamagnetic iron oxide nanoparticles coated with an alginate are used in combination with a free alginate.

15. The composition of claim 13, the plurality of superparamagnetic iron oxide nanoparticles coated with an alginate or the use of claim 14, wherein a concentration of said free alginate ranges from 0.02 to 0.06 percent by weight of the total weight of said sample.

16. The composition, plurality of superparamagnetic iron oxide nanoparticles coated with an alginate or use of claim 15, wherein said magnetic resonance imaging or spectroscopy is used for determining a presence and/or level of calcium ions in a range of from 2 mM to 5 mM.

17. The composition of claim 13, the plurality of superparamagnetic iron oxide nanoparticles coated with an alginate or the use of claim 14, wherein a concentration of said free alginate ranges from 0.002 to 0.02 percent by weight of the total weight of said sample.

18. The composition, plurality of superparamagnetic iron oxide nanoparticles coated with an alginate or the use of claim 17, wherein said magnetic resonance imaging or spectroscopy is used for determining a presence and/or level of calcium ions in a range of from 0.2 mM to 2 mM.

19. The method of any of claims 1-7, the composition of any of claims 9, 12,

13, 15, 16, 17 or 18, the plurality of superparamagnetic iron oxide nanoparticles coated with an alginate of any of claims 10, 12, 14, 15 or 17, or the use of any of claims 11, 12,

14, 15, 17 or 18, wherein the sample comprises an aqueous medium.

20. The method, composition, plurality of superparamagnetic iron oxide nanoparticles coated with an alginate or use of claim 19, wherein the sample is selected from the group consisting of an aqueous solution, a biological sample, a bodily fluid sample, a cell culture, a plant sample, a tissue sample, an organ sample and a bodily site.

21. The composition of any of claims 9, 12, 13, 15, 16, 17 or 18, the plurality of superparamagnetic iron oxide nanoparticles coated with an alginate of any of claims 10, 12, 14, 15 or 17, or the use of any of claims 11, 12, 14, 15, 17 and 18, wherein said determining is effected is the presence of ions of a metal other than calcium.

22. A method of monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject, the method comprising:

administering to the subject a diagnostically effective amount of a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate;

subjecting the subject to magnetic resonance imaging or spectroscopy;

and detecting a formation of aggregates of at least a portion of said superparamagnetic iron oxide nanoparticles coated with said alginate, wherein a formation and/or level of formation of said aggregates is indicative of the presence and/or level of calcium ions in an organ or tissue of the subject,

thereby monitoring the presence and/or progress of the medical condition.

23. Use of a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate in the manufacture of a diagnostic agent for monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject by magnetic resonance imaging or spectroscopy.

24. A plurality of superparamagnetic iron oxide nanoparticles coated with an alginate for use in monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject by magnetic resonance imaging or spectroscopy.

25. A pharmaceutical composition comprising a plurality of superparamagnetic iron oxide nanoparticles coated with an alginate, the composition being identified for use in monitoring a presence and/or progress of a medical condition associated with a change in a level of calcium ions in a subject by magnetic resonance imaging or spectroscopy.

26. The method, use, plurality of superparamagnetic iron oxide nanoparticles coated with an alginate or composition of any of claims 22-25, wherein said medical condition is selected from the group consisting of hypocalcaemia, hypercalcaemia, constipation, eating disorders, chronic renal failure, severe acute hyperphosphatemia, ineffective PTH syndroms, pancreatitis, alkalosis, bone pain, kidney stones, psychotic behavior, depression, confusion, psychiatric overtones, cerebral ischemia, cardiac ischemia, cardiac arrest, heart failure, cancer, neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson, multiple sclerosis and Alzheimer disease.

27. The method, use, plurality of superparamagnetic iron oxide nanoparticles coated with an alginate or composition of any of claims 22-25, wherein said level of calcium ions in a bodily site of said subject ranges from 100 μΜ to 5 mM.

28. The method of claim 22, further comprising administering to the subject an effective amount of a free alginate.

29. The use of claim 23, wherein said diagnostic agent is used in combination with a free alginate.

30. The plurality of superparamagnetic iron oxide nanoparticles coated with an alginate of claim 24, for use in combination with a free alginate.

31. The composition of claim 25, further comprising a free alginate.

32. The composition of claim 25, for use in combination with a free alginate.