Superparamagnetic Iron Oxide Nanoparticles

8/9/2019 Superparamagnetic Iron Oxide Nanoparticles

1/21

Review Article

Superparamagnetic iron oxide nanoparticles:diagnostic magnetic resonance imaging and

potential therapeutic applications inneurooncology and central nervous systeminflammatory pathologies, a review

Jason S Weinstein1, Csanad G Varallyay2,3, Edit Dosa2, Seymur Gahramanov2,Bronwyn Hamilton4, William D Rooney5, Leslie L Muldoon2 and Edward A Neuwelt1,2,6

1Department of Neurological Surgery, Oregon Health and Science University, Portland, Oregon, USA;2Department of Neurology, Oregon Health and Science University, Portland, Oregon, USA; 3Department ofNeuroradiology, Universitatsklinikum Wurzburg, Wurzburg, Germany; 4Department of Radiology, Oregon

Health and Science University, Portland, Oregon, USA; 5

Advanced Imaging Research Center, OregonHealth and Science University, Portland, Oregon, USA; 6Portland Veterans Affairs Medical Center,Portland, Oregon, USA

Superparamagnetic iron oxide nanoparticles have diverse diagnostic and potential therapeuticapplications in the central nervous system (CNS). They are useful as magnetic resonance imaging(MRI) contrast agents to evaluate: areas of bloodbrain barrier (BBB) dysfunction related to tumorsand other neuroinflammatory pathologies, the cerebrovasculature using perfusion-weighted MRIsequences, and in vivo cellular tracking in CNS disease or injury. Novel, targeted, nanoparticlesynthesis strategies will allow for a rapidly expanding range of applications in patients with braintumors, cerebral ischemia or stroke, carotid atherosclerosis, multiple sclerosis, traumatic braininjury, and epilepsy. These strategies may ultimately improve disease detection, therapeuticmonitoring, and treatment efficacy especially in the context of antiangiogenic chemotherapy andantiinflammatory medications. The purpose of this review is to outline the current status ofsuperparamagnetic iron oxide nanoparticles in the context of biomedical nanotechnology as theyapply to diagnostic MRI and potential therapeutic applications in neurooncology and other CNSinflammatory conditions.Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1535; doi:10.1038/jcbfm.2009.192; published online16 September 2009

Keywords: bloodbrain barrier; CNS tumors; magnetic resonance imaging; ultrasmall superparamagnetic ironoxide nanoparticles

Introduction

The diagnosis and treatment of pathologies thataffect the central nervous system (CNS) are currentlyundergoing a renaissance because of the markedproliferation of nanoscale technologies. Nanotech-nology, as it relates to biomedicine, can broadly bedefined as nano-sized structures that contain at least

one dimension between 1 to 100nm in sizeyandpossess new or enhanced properties that are un-

attainable at both smaller (quantum) [and] larger(macromolecular) levels (Hartmanet al, 2008).

Superparamagnetic iron oxide nanoparticles arebased on magnetite (Fe3O4), which has received themost attention for biomedical applications, or ma-ghemite (gFe2O3) molecules encased in polysacchar-ide, synthetic polymers, or monomer coatings(Laurent et al, 2008;Thorek et al, 2006). The utilityof superparamagnetic iron oxides as magnetic reso-nance imaging (MRI) contrast agents has beenstudied for more than two decades (Weisslederet al , 1990) and the list of available agents israpidly expanding (Table 1). These particles can be

Received 3 April 2009; revised 11 August 2009; accepted 13August 2009; published online 16 September 2009

Correspondence: Dr EA Neuwelt, Department of NeurologicalSurgery, Oregon Health and Science University, 3181 SW SamJackson Park Road, L603, Portland, OR 97239, USA.E-mail:[email protected]

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1535

&2010 ISCBFM All rights reserved 0271-678X/10 $32.00

www.jcbfm.com
http://dx.doi.org/10.1038/jcbfm.2009.192mailto:[email protected]://www.jcbfm.com/http://www.jcbfm.com/mailto:[email protected]://dx.doi.org/10.1038/jcbfm.2009.192


2/21

organized according to their hydrodynamic diameterinto several categories (Corot et al, 2006): standardsuperparamagnetic iron oxide particles (SPIOs) (50to 180 nm), ultrasmall superparamagnetic iron oxideparticles (USPIOs) (10 to 50 nm), and very smallsuperparamagnetic iron oxide particles (VSPIOs)( < 10 nm). Most contemporary investigations useUSPIOs; therefore, for the sake of consistency wewill refer to superparamagnetic iron oxide nanopar-ticles, in general, as USPIOs unless specifically

discussing SPIOs or VSPIOs.Particles of iron oxide have been administeredintravenously (IV) for over 50 years, initially for thetreatment of anemia (Cameronet al, 1951). Emergingexperimental and clinical applications in the CNScapitalize on both the physical and magnetic proper-ties of iron oxide nanoparticles; the list of biomedicalimaging applications for these nanoparticles con-tinues to expand. There are a number of importantqualities of USPIOs that make them attractive ascomplimentary or alternative MRI contrast agentscompared with gadolinium-based contrast agents(GBCAs). These can be summarized as follows:USPIOs are virus-sized molecules with a very long

circulating half-life (B14 h for ferumoxytol); USPIOsare avidly taken up by phagocytic cells such as theKupffer cell fraction of the liver, circulating mono-cytes/macrophages and mononuclear T cells, as wellas reactive astrocytes, microglia, and dendritic cellswithin the brain. Neutrophils have not been found totake up USPIO. The USPIOs are cleared from thecirculation primarily by the reticuloendothelialsystem (Bourrinet et al, 2006); these properties (i.e.,degree of cellular labeling and rate of clearance) aredependent on size, coating, and method of deliveryand will be discussed in detail below. Limitations ofthese agents for current CNS imaging applications

are primarily related to the inability to reliablydifferentiate USPIO signal from resident brain ironsignal (i.e., in the setting of hemorrhage related tostroke or trauma). There are also very little data inhumans regarding the long-term clearance of theseagents from the brain.

Ferumoxides (Endorem, Guerbet in Europe; Fer-idex, AMAG Pharmaceuticals Inc in the USA andJapan) and ferucarbotran (Resovist, Schering Bayer inEurope and Japan) are commercially available SPIOs

approved for MR imaging of liver tumors (Ros et al,1995). Clinical CNS imaging studies have also beenperformed with these compounds (Rose et al, 2006;Varallyay et al , 2002). The USPIO, ferumoxytol(Feraheme, AMAG Pharmaceuticals Inc, CambridgeMA, USA) is approved for iron-replacement therapyin patients with chronic renal failure (Kidney Daily,6/30/2009). Ferumoxtran-10 (Manninger et al, 2005;Salehet al, 2007), SHU555C (Vellingaet al, 2008), andferumoxytol (Neuwelt et al, 2007) have been inves-tigated in humans for various CNS imaging applica-tions. Preliminary studies using ferumoxides, feru-moxtran-10 (Combidex), and ferumoxytol have notrevealed significant toxicities (Muldoon et al, 2006;

Neuwelt et al, 2007). Ferumoxytol, in particular, isattractive as an MRI contrast agent because it can begiven as a bolus for first-pass perfusion imaging andappears to be safe in patients with chronic kidneydisease; at later time points (i.e., 24 h), ferumoxytolaccumulation is evident in areas of bloodbrainbarrier (BBB) dysfunction that may be fundamentallyrelated to inflammation from any cause.

This review will focus primarily on USPIOs,specifically ferumoxtran-10 and ferumoxytol, fordiagnostic applications in the CNS with an emphasison their utility as MRI contrast agents in the settingof CNS tumors. It will also highlight the utility of

Table 1 Available superparamagnetic iron oxide agents and prohance (Gd-based agent) for comparison

Name Developer Coating agent Size (nm)a Clinical dose(mmol Fe/kg)

Relaxivity(mM1 sec1)b

Ferumoxides AMI-25Feridex/Endorem

Guerbet AMAGPharm. Inc

Dextran T10 120180 (SPIO) 30 r1 =10.1r2 =120

Ferucarbotran SH U 555 A

Resovist

Bayer Schering

Pharma AG

Carboxydextran 60 (SPIO) 812 r1 =9.7

r2 =189Ferumoxtran-10 AMI-227Combidex/Sinerem

GuerbetAMAG Pharm. Inc

Dextran T10, T1 1530 (USPIO) 45 r1 =9.9r2 = 65

Ferumoxytol Code 7228 AMAG Pharm. Inc Polyglucose sorbitolcarboxymethyl ether

30 (USPIO) 1874 r1 = 15r2 = 89

SH U 555 C Supravist Bayer ScheringPharma AG

Carboxydextran 21 (USPIO) 40 r1 =10.7r2 = 38

Feruglose NC-100150Clariscan

GE-Healthcare Pegylated starch 20 (USPIO) 36 n.a.

VSOP-C184 Ferropharm Citrate 7 (VSPIO) 1575 r1 = 14r2 =33.4

Gadoteridol (ProHance) Bracco Diagnostics, Inc 1 (GBCA) 100 (umol (Gd) /kg) r1 = 4r2 = 6

Currently available intravenous iron oxide nanoparticle contrast agents. Modified from Corot et al (2006).a

Hydrodynamic diameter, laser light scattering.b

Relaxometric properties (mM1

sec1

) at 1.5T, 37 1C, water or in plasma; per mM Gd, or Fe.

Superparamagnetic iron oxide nanoparticles in brainJS Weinstein et al

rnal of Cerebral Blood Flow & Metabolism (2010) 30, 15 35


3/21

USPIOs for imaging tumor neovasculature andassessing therapeutic response to antiangiogenicchemotherapeutic agents.

Basic science review

Ultrasmall Superparamagnetic Iron Oxide ParticleSynthesis and Pharmacology

The most common methods of USPIO synthesis forbiomedical applications are coprecipitation of ferricand ferrous salts in an alkaline medium, with orwithout surface complexing agents such as dextranor polyethylene glycol, or microemulsion techniqueswith small amounts of iron ions trapped insidesurfactant bubbles in an oil medium (Hartman et al,2008;Laurentet al, 2008). The USPIOs consist of twocomponents: an iron oxide core and a hydrophiliccoating. It is the combination of these two compo-nents that determines their pharmacology. Passivetargeting is perhaps most dependent on the hydro-dynamic radius and the surface charge (factorsrelated to the coating material), as these character-istics determine circulation time, accessibility totissues, opsonization, and rate of cell-type uptake(Thorek et al, 2006). Active targeting in the CNStakes advantage of nanoparticle surface modifica-tions (e.g., the addition of monoclonal antibodies orpeptides) such as chlorotoxin, for glioma imaging.Current clinical trials primarily involve passivetargeting.

With respect to passive targeting, USPIOs behavedifferently than GBCAs for a number of reasons.

First, because of their larger molecular sizeup to50 nm (compared with the 1 nm gadolinium chelate),USPIOs extravasate much more slowly than standardGBCA, even in areas of severe BBB dysfunction (i.e.,malignant glioma). Most GBCAs, in comparison,rapidly extravasate into the extravascular space inareas of BBB dysfunction and are rapidly clearedfrom the circulation via the kidneys. There arenumerous formulations based on the gadoliniumion [Gd(III)], each with unique properties and safetyprofiles (Harpur et al, 1993).

Ferumoxtran-10 is a first-generation USPIO thatmay have a slight advantage over ferumoxtyol foranatomic imaging of inflammatory lesions; however,

it is not safe for angiographic imaging procedures,which require a rapid infusion. Ferumoxytol wasdeveloped as an IV iron-replacement therapy, speci-fically for anemic patients with chronic kidneydisease; after bolus IV administration, it follows auseful distribution that is conceptionally based onthe conservation of mass. Over time, a combinationof events occur leading to contrast enhancement:USPIOs slowly leak across the BBB (mechanismincompletely understood); there may also be uptakeof USPIO by circulating monocytes/macrophage,which then cross the BBB in response to inflamma-tion and injury. Histologic samples from resected

brain tumors of patients, who received USPIOs,reveal that part of the extravasated iron oxideparticles are taken up by parenchymal cells. Thus,their localization is both intracellular and inter-stitial (Figure 1A) (Varallyay et al, 2002).

Measurable T1-weighted signal enhancement (hy-perintensity) in brain tumors is seen as early as 4 to

6 h after USPIO injection; contrast enhancementintensity peaks B24 h after injection and cangenerally be visualized even 72 h after injection,although it is usually faint and more diffuse(Neuwelt et al, 2007). In contrast to GBCAs, there isno renal elimination of USPIO, which accounts forthe enhanced safety profile in patients with renaldysfunction who appear to be at increased risk forcontrast-induced nephropathy or nephrogenic sys-temic fibrosis (Neuwelt et al, 2008).

Unlike USPIOs, the plasma half-life of SPIOs is onthe order of minutes. This is due to their largerparticle size ( > 50 nm) and negative surface charge,which lead to rapid elimination. Iron-loaded mono-nuclear cells can actively cross a relatively intactBBB in cases of an active inflammatory process(Oude Engberink et al, 2007), but because of theirlarge size, SPIOs do not leak across BBB defectslike free USPIOs (as in malignant gliomas). In theclinical setting, FDA-approved doses of ferumoxides(SPIOs) were given to 20 patients with intrinsicor metastatic brain tumors. No enhancement wasvisualized in any patient at 30 mins or 4 h (Varallyayet al, 2002).

The safety profile of clinically useful USPIOsvaries based on their molecular structure. Aftercellular internalization, iron oxide nanoparticles

accumulate in lysosomes in which the low pH breaksthe iron oxide core down into iron ions. These ionsare then incorporated back into the hemoglobin pool(Thorek et al, 2006). The type of coating used has asignificant impact on the immunologic response toUSPIOs; to date, studies have shown that polymer-coated nanoparticles have minimal impact on cellviability and function (Bourrinet et al, 2006;Thoreket al, 2006). Trypan blue exclusion-based toxicitystudies, using high concentrations of USPIOs, showgood tolerance with minimal cell death. However,there have been reports of free radical generation,decreased cell proliferation, and even cell death withsome formulations, highlighting the uniqueness of

different nanoparticle configurations (Modo et al,2005). Whereas earlier agents, such as ferumoxtran-10, were administered via slow infusion to avoidmast cell degranulation, newer agents, such as SHU555 C and ferumoxytol can be safely given as a rapidIV bolus.

Ferumoxytol has been tested as an iron supple-ment therapy in patients with renal failure up to thedose of 510 mg (Landry et al, 2005;Spinowitz et al,2005). For MRI contrast agent applications, theamount of iron administered (typically < 4 mg/kg)is much lower than doses resulting in acute systemictoxicity (above 60 mg/kg) or chronic iron overload


1

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 15 3


4/21

(above 20 g stored iron). Over 1700 patients havereceived ferumoxytol in its clinical developmentprogram, and at least 1500 of these were patientswith chronic kidney disease in Phase 3 iron-replace-ment studies. To date, only one patient, with ahistory of multiple drug allergies, has experienced ananaphylactoid reaction (hot flashes and itching,without respiratory compromise) and severe hypo-tension a few minutes after receiving ferumoxytol.There have been no deaths that were considered to

be related to ferumoxytol treatment (Neuwelt et al,2008). The long-term fate of iron from ferumoxytol inthe brain remains unknown, however. There isgrowing evidence that iron accumulation in thebrain, as in nigrostriatal dopaminergic neuronsassociated with Parkinsons disease for example, isnot merely an epiphenomenon but a result ofmisregulation of iron homeostasis within the brain(Ke and Ming Qian, 2003). Preclinical studies ofUSPIOs injected directly into the rat brain, ordelivered transvascularly, have not revealed anyacute or midterm toxicity. As early as 7 days aftertransvascular delivery of ferumoxytol, no iron was

detectable in the brain using MRI and histology(Muldoon et al , 2005). After direct intracerebralinjection of USPIO or SPIO, iron was still easilydetected in brain parenchyma by MRI and histology,with no sign of pathology at 3 months.

Magnetic Resonance Imaging Physics

Magnetic resonance imaging is a noninvasive tech-

nique that uses magnetic fields to produce highresolution and high-contrast images of tissue struc-ture and function. The principal tissue signal ofinterest in essentially all clinical MRI arises fromwater protons. Water concentration can vary signifi-cantly between biological tissues and this property isexploited to produce a fundamental contrast in MRIthat is known as proton density contrast. In protondensity-weighted MRI, the signal intensity of eachvoxel is proportional to the local proton concentra-tion. Another fundamental class of MRI contrastrelies on spatial differences in the relaxation proper-ties of the MR signal.

Figure 1 (A) 18: Adapted with permission from Varallyay et al (2002). Patient with anaplastic oligodendroglioma. A.12:nonenhanced (A.1) and gadolinium-enhanced (A.2) SE T1-weighted images of a right temporal tumor. The gadolinium-enhancedimage shows strong, lobulated peripheral enhancement with a central nonenhancing region. A.3: at 24 h after ferumoxtran-10(Combidex) infusion, SE T1-weighted image shows high-signal intensity in a similar distribution, but with less peripheral lobulation,compared with the gadolinium-enhanced image. Also note that the nongadolinium-enhancing central zone became isointense towhite matter, suggesting some ferumoxtran accumulation. A.46: fast SE T2-weighted image obtained before ferumoxtran-10infusion (A.4) and fast SE T2-weighted (A.5) and GRE T2*-weighted (A.6) images obtained 24 h after ferumoxtran-10 infusion showa heterogeneous tumor mass with peripheral decreased signal intensity that is more prominent on the GRE T2*-weighted image. Thedistribution of low-signal intensity is similar to that of the high-signal intensity areas on the SE T 1-weighted image (A.3). A.78:photomicrographs of pathologic specimens stained for iron (DAB-enhanced Perls stain). A.7: (original magnification7.5 ; barindicates 1 mm), tumor (T) and reactive brain interface (RB) show intense staining for iron at the periphery of the tumor. A.8: (originalmagnification100 ; bar indicates 0.1mm), cellular iron staining at the tumor-reactive brain interface shows iron uptake byparenchymal cells with fibrillar processes (arrows) rather than within the round tumor cells (T) themselves. (B) Adapted withpermission fromMuldoon et al (2005). Iron particle imaging of human small-cell lung cancer (LX-1 cell line) xenografts in a rodentmodel. B.18: T1 MRI scans (1.5T) comparing ferumoxtran-10 (top), ferumoxytol (middle), and ferumoxides (bottom) 24 h afterintravenous administration in nude rats with LX-1 intracerebral tumors. All animals were imaged at 9 days after tumor cell

implantation except #3 and #4, which were imaged on day 6. B.910: iron histochemistry photomicrographs. Intense iron stainingwas found at the LX-1 tumor/reactive brain interface 24 h after administration of ferumoxtran-10 or ferumoxytol.




5/21

There are two principal relaxation processes thatcharacterize MR signals, one that relates how rapidlymagnetization parallel to the strong static magneticfield recovers after a perturbation, and the other thatdescribes how rapidly magnetization transverse tothe static magnetic field decays after it has beenproduced by a series of radiofrequency pulses. The

constants that characterize these two kinetic pro-cesses are referred to as longitudinal and transverserelaxation time constants, T1, and T2, respectively. Ingeneral, T1 relaxation processes are sensitive tofluctuating magnetic fields at or about the MRIoperational frequency (i.e., the Larmor frequency).Transverse relaxation processes are sensitive tomagnetic fields that fluctuate at the Larmor fre-quency, but also are significantly determined byfluctuations at low frequency. In complex samplessuch as tissues, the density of low-frequencyfluctuating fields is much greater than those at ornear the Larmor frequency. This is why 1H2O T2values are typically much smaller than T1 values intissue. An apparent transverse relaxation time con-stant (T2*) can also be defined. In addition tocontributions from T2 mechanisms, T2* relaxationprocesses are sensitive to microscopic magnetic fielddistributions. Such magnetic field distributions canbe produced by compartmental differences in mag-netic susceptibility, such as occurs in areas proximalto blood vessels with significant deoxyhemoglobinconcentration and is exploited in functional MRIexperiments. Therefore, T2* values are alwayssmaller than T2 values.

For most MRI studies, the intrinsic contrastprovided by spatial differences in proton density

and relaxation times is sufficient. However, it isimportant to appreciate that most MRI acquisitions,although strongly weighted to a particular type ofcontrast, are invariably sensitive to more than onetype of contrast. The amount of mixed weighting inan MRI acquisition depends on a number of factors,but becomes important when discussing contrastagent applications.

Magnetic Resonance Imaging Contrast Agents

Although intrinsic contrast is sufficient for most MRIapplications, exogenous contrast agents are used inB40% of all clinical MRI studies. Typically, theseagents are used to increase lesion conspicuity and toimprove characterization of blood vessels. Unliketracers used in x-ray or nuclear medicine imaging,MRI contrast agents are detected indirectly throughtheir ability to catalyze water proton relaxation andperturb MRI signal intensity. By far, the most widelyused MRI contrast agents are those based on theparamagnetic gadolinium [Gd(III)] ion. The Gd(III)ion has seven unpaired electrons and favorableelectronic spin relaxation properties that make forvery efficient catalysis of water proton relaxation.The Gd(III) ion is chelated to a low-molecular weight

ligand to reduce toxicity. These low-molecularweight GBCAs are injected IV, most will distributerapidly into all accessible extracellular spaces, andare eliminated from the body through the kidneyswith a typical elimination half-life of B1.6h. Con-trast agents catalyze relaxation rate constants (theinverse of the time constants described above) in a

concentration-dependent manner. In simple solu-tions, the 1H2O T1

1 increases linearly with contrastagent concentration. The slope of this dependence isknown as the relaxivity, typically reported in units ofmmol1 sec1, and is a measure of how potent theagent is for catalyzing relaxation. Relaxivities typi-cally differ for longitudinal and transverse relaxationand vary with magnetic field strength. The GBCAstypically are used in combination with T1-weightedMRI acquisitions and produce a hyperintense(bright) signal in tissue regions in which the agentaccumulates. The low-molecular weight and weakprotein binding characteristics of most GBCAs leadto avid extravasation of GBCAs from normal bloodvessels in most tissue and abnormal blood vessels inthe CNS. These agents have found widespread usefor investigations of bloodCNS barrier disruptionfound in many disease pathologies.

The USPIOs are based on magnetite (Fe3O4)nanocrystals and are classified as superparamagneticcompounds because the net magnetic dipole momentrealized exceeds that expected from the unpaired[Fe(II), Fe(III)] electrons alone. Like GBCAs, theUSPIOs do not retain any net magnetism onceremoved from the strong magnetic field; thermalenergy is sufficient to destroy the net magnetic orderwithin the nanocrystal established by the strong

magnetic field. The USPIOs have excellent relaxiv-ities and on a per iron-atom basis compare veryfavorably with GBCAs (Table 1). Unlike the GBCAs,which have similar transverse and longitudinalrelaxivities at clinically relevant magnetic fields,the USPIOs have significantly greater transverserelaxivities compared with longitudinal relaxivities(Table 1). Thus, USPIOs tend to find greater applica-tion on magnetic susceptibility-based acquisitions inT2-weighted or T2*-weighted MRI, in which theyproduce a hypointense (dark) signal (Figure 2A). Thestrong magnetic susceptibilities of these compoundscan result in a significant distribution of microscopicmagnetic fields and severe MR signal quenching.

This can be appreciated inFigure 2Athat comparesminimum intensity projection susceptibility-weighted images obtained without exogeneous con-trast, with a standard dose GBCA, and with 4 mg/kgferumoxytol. The image collected after ferumoxytolshows significantly greater MR signal quenching(hypointensity) in areas in and surrounding bloodvessels than either the GBCA or no exogeneouscontrast agent conditions. Nevertheless, USPIOs dohave significant longitudinal relaxivities and havebeen used as agents in T1-weighted and evendynamic contrast-enhanced acquisitions, in whichtissue regions that accumulate the agent appear


1



6/21

hyperintense. It should be noted that the transverserelaxivities increase supralinearly with magneticfield strength, whereas the longitudinal relaxivitiestypically decrease slightly (Blockley et al, 2008).Therefore, the hyperintense T1-weighted enhance-ment is more readily achieved at lower magneticfield strengths and the potential for mixed weightingeffects in USPIO-based MRI applications increasesmarkedly with magnetic field strength (Muldoonet al , 2005; Neuwelt et al , 1994). Comparingpostcontrast T1-enhancement at 3 T and 7 T in thesame subject at similar times reveals improved

sensitivity for GBCA detection but reduced sensitiv-ity for USPIO detection at 7 T (Figure 2B). Theobservation of increasing GBCA detection sensitivitywith magnetic field is consistent with predictionsbased on increased nominal brain tissue T1 relaxa-tion time constants with increasing magnetic field(Rooney et al, 2007). Increased nominal T1 valuesimparts increased sensitivity for detecting lowerconcentration of GBCA despite a slight decrease inlongitudinal relaxivity and increase in transverserelaxivity with increasing magnetic field (Changet al, 1994;Rohrer et al, 2005).

no contrast

1

GBCA

2

3

ferumoxytol

4

21

3 4 5 6 7

5

60

50

40

30

20

10

00 10 20 30 40 50 60 70 80

Time (Hours)

Signal Intensity curves on

1.5 and 3 Tesla

1.5 Tesla

NormallzedSignal

Intensity

6

321

3T

7T

no contrast GBCA ferumoxytol

3 Tesla

Figure 2 (A) MRI showing cerebral vasculature at 7 T. Minimum intensity projection (mIP) susceptibility-weighted images wereobtained without contrast agent (A.1), with GBCA (gadoteridol, 0.1 mmol/kg, A.2), and immediately after USPIO injection(ferumoxytol 510 mg, A.3). The USPIO enhancement results in superior visualization of small intracerebral vessels. (B) 3D MPRAGET1-weighted MRI in a patient at 3 T and 7 T without contrast (B.1 and B.4), with GBCA (B.2 and B.5), and with ferumoxytol(510 mg, 24 h after infusionB.3 and B.6). GBCA associated T1-weighted signal intensity increases at higher magnetic fieldstrength because of the increased nominal brain tissue T1 relaxation time constant, whereas ferumoxytol enhancement signalintensity is reduced because of increased T2* contributions at 7 T. (C) Adapted with permission from Neuwelt et al (2007). Timecourse of ferumoxytol enhancement in a patient with recurrent GBM. GBCA-enhanced T1-weighted (C.1), T2-weighted (C.2), andpost-ferumoxytol T1-weighted (C.37) MRI scans were obtained using a 1.5 T MRI at five time points (C.3 = 46 h; C.4 = 620h;C.5 = 2428 h; C.6 = 4852 h; C.7 =B72h). Peak intensity is observed at the 24 to 28h time point; much later than thatobserved with GBCA (not shown), which enhances maximally 3.5 to 25mins after injection. The figure shows comparison offerumoxytol-enhancement intensity curves at 1.5 T and 3 T MRI. Maximum ferumoxytol T1enhancement is greater at 1.5T than on 3 T.




7/21

Generally, ther1/r2 ratios decrease with increasingfield strength and these alterations are especiallypronounced for USPIOs (Rohrer et al, 2005). Hence,USPIOs show superior T1-weighted contrast at lowermagnetic field strengths, as in a 0.15 T intraoperativeMRI (Hunt et al, 2005), using standard T1-weightedsequences. A comparison of 1.5 T versus 3 T scans,

with analogous imaging sequences, using ferumox-ytol in patients with CNS malignancies revealed thatturbo spin echo (TSE), a rapid T1-weighted sequence,at 1.5 T resulted in greater changes in signal intensitythan TSE sequences at 3T (Neuwelt et al, 2007).Furthermore, similar patient groups have beenstudied at 3 T versus 7 T; the lower magnetic fieldprovided a larger area of signal enhancement onT1-weighted magnetic prepared rapid gradient echo(MPRAGE) MRI (unpublished data; Figure 2B). At12T, no T1-weighted enhancement was found withUSPIOs in preclinical tumor models (unpublisheddata). Newer imaging sequences, such as Ultrashortecho time sequences (Idiyatullin et al, 2006), andInversion-Recovery with ON Resonant Water Sup-pression, open new avenues for obtaining hyper-intense enhancement with USPIOs (Korosoglouet al,2008; Stuber et al , 2007). However, thus far noapplications in CNS imaging have been reported.

Diagnostic and potential therapeuticapplications

Imaging Central Nervous System Tumors withUltrasmall Superparamagnetic Iron Oxide Particles

There areB

16,900 new cases of primary CNS tumorsdiagnosed in the United States each year. Contrast-enhanced MRI of CNS malignancies is a crucialdiagnostic tool and a key parameter for follow-upimaging of tumor response to therapy (Macdonaldet al, 1990). Enhancement of residual or recurrenttumors using standard GBCAs reflect both cerebralblood flow (CBF) and alterations in the BBB appear-ing as increased signal on T1-weighted images withinminutes after injection (Akeson et al, 1997). UnlikeGBCAs, USPIO enhancement at early time pointsafter administration is not a clear marker of BBBdeficiency. The mechanism of USPIO tissue accu-mulation is not fully understood but is likely related

to its prolonged circulation time and as a cellular labelfollowing uptake by inflammatory cells within andaround tumors. For CNS tumor imaging, T1-weightedscans have proven to be superior for the evaluation oflow concentrations of iron oxide nanoparticles traver-sing the BBB (Muldoon et al, 2005; Neuwelt et al,1994; Varallyay et al, 2002). But as discussed above,this observation is dependent on details of the MRIacquisition including the magnetic field strength.

The BBB is a special feature of CNS capillaries thatresults from a continuous layer of endothelial cellsbound together with tight junctions that allow verylittle transcellular or pericellular transport of blood

borne molecules (Banks, 1999). Micrometastases thatlack neovascularity remain protected by the BBB andmay be undetectable using GBCAs. In contrast to thenormal BBB, the bloodtumor barrier (BTB) may behighly permeable and allows conventional contrastagents to leak from the vessels into the perivascularspace (Groothuis et al , 1991; Long, 1979). The

permeability of the BTB may be variable not onlyin different histologic types of tumors but also withinone tumor mass (Barnett et al, 1995; Kraemer et al,2001;Kroll and Neuwelt, 1998;Varallyayet al, 2002),increasing the difficulty of accurately determiningtumor size and type. Furthermore, any of a variety ofinflammatory or infectious CNS lesions may showsimilar patterns of GBCA enhancement complicatingthe differential diagnosis (Enochs et al, 1999).

The USPIOs, which can be used both as intravas-cular contrast agents and as a cellular imaging agent,may be useful in the reduction of the above-mentioned problems (Corot et al, 2004). In animalstudies, ferumoxtran-10 shows slightly better tumorimaging at the same dose as ferumoxytol; however, itmust be administered over B30 mins to limit adversereactions (Figure 1B) (Neuweltet al, 2004). Enhance-ment after IV infusion of ferumoxtran-10 increasesslowly and peaks at B24 h after administration; thendeclines during the next several days (Figure 2C)(Manninger et al , 2005; Neuwelt et al , 2004;Varallyay et al, 2002). Indeed, after resection, ourgroup found that residual lesions were still readilyvisible on the T1-weighted postoperative MRI at 48 h.This property of ferumoxtran-10 allows for assess-ment of residual tumor without the need to re-administer a contrast material during intraoperative

MRI (0.15 T) or on postoperative MRI (Hunt et al,2005). In additional clinical trials, three patientswith glioblastoma multiforme (GBM) who receivedearlier radiation showed fewer areas of enhancementwith ferumoxytol than with GBCA. When comparedwith ferumoxtran-10, ferumoxytol seemed to providesomewhat less enhancement. In another study, 6 outof 14 patients with GBM had MRIs, which revealedmore intense enhancement with ferumoxtran-10than with GBCA, whereas our group found 1 patientout of 5 with GBM had an MRI showing slightlyincreased enhancement with ferumoxytol versusGBCA (Neuwelt et al, 2007).

In an attempt to improve diagnostic specificity,

multifunctional modifications are increasingly usedto more specifically target USPIOs to the intendedtarget. One example of this involves attachment ofchlorotoxin, a 4-kDa peptide purified from theLeiurus quinquestriatus scorpion. It is a highlyspecific marker for glioma cells in biopsy tissues(Soroceanu et al, 1998) that can target tumors inanimal models (Lyonset al, 2002).

Early clinical applications of USPIOs as MRIcontrast agents focused on investigations of bloodCNS barrier breakdown in neuroinflammation andneoplasm. More recently, these agents have foundwidespread use in dynamic MRI examinations,


2



8/21

including dynamic susceptibility contrast (DSC). TheDSC techniques are useful for quantifying tissueperfusion (blood volume and blood flow) and areaccomplished with time-series T2*-weighted MRIdata that are collected during the bolus administra-tion of a contrast agent. Dynamic contrast enhance-ment measurements are collected using a T1-

weighted MRI time series, and are useful forquantifying contrast agents vascular permeabilityand tissue distribution volumes. Early experimentaldata suggest that the combination of GBCA-enhancedT1-weighted MRI and USPIO-enhanced T2-weightedMRI reveals complimentary information in patientswith CNS tumors (Neuwelt et al, 2007).

Perfusion-weighted imaging (PWI) is generallyperformed using first-pass, dynamic susceptibility-weighted contrast-enhanced (DSC) MRI echo-planarimaging approaches (Cha et al, 2002). The basicprinciple of PWI using DSC MRI is as follows: thefirst-pass effect of a contrast bolus in brain tissue ismonitored using a series of T2*-weighted MR imagesfrom the same brain regions, which are scannedrepeatedly. The susceptibility effect of a paramag-netic (GBCA) or superparamagnetic (USPIO) contrastagent causes signal loss that can be converted into acontrast agent concentration (Essig et al, 2006).

From these data, parametric maps of CBF, cerebralblood volume (CBV), and mean transit time (MTT =CBV/CBF) are calculated in regions of interest(Ostergaard, 2005). The CBF can be defined as theamount of blood delivered to a standard volume ofbrain per unit time, such as 50 mL/100 gm/mins ingray matter. The CBV is the amount of blood pervolume of brain or pathologic lesion. The MTT refers

to the time it takes for a bolus of contrast to passthrough a region of tissue; it can be calculated bycomparing a signal washout curve in the region ofinterest with the contrast signal in a cerebral artery(Figure 3A). These data parameters, alone or incombination, provide information on cerebral hemo-dynamics and serve as surrogates to quantify angio-genesis. The USPIOs may provide more accuratemeasurements of these vascular parameters, com-pared with GBCAs, because of their propensity toremain intravascular at early time points (Figure 3B).

Molecular Imaging of Inflammation with UltrasmallSuperparamagnetic Iron Oxide Particles

Molecular imaging is an important new diagnostictool for studying in vivo cellular and molecularbiology across a wide range of disciplines. Molecularimaging, outlined as the noninvasive, quantitative,and repetitive imaging of targeted macromoleculesand biological processes in living organisms(Herschman, 2003) could allow for earlier diseasedetection; more accurate prognostic information andpersonalized treatment strategies; an enhanced abil-ity to monitor the efficacy of treatment; and animproved understanding of how cells behave and

interact in their microenvironment in vivo(Thoreketal, 2006). There are a number of interesting investi-gations being conducted using SPIO-labeled dendri-tic cells in the setting of neurooncology (Verdijket al,2007) as well as molecular imaging of inflammationand stem cell tracking using GBCAs and otherbioactive agents such as fluorine (Brekke et al ,

2007; Ruiz-Cabello et al, 2008). The field of mole-cular imaging is expanding rapidly and is beyond thescope of this review; however, we will touch on someof the studies completed by our group and othersusing USPIOs.

Phagocytic cellular uptake of iron oxides increaseswith particle size (Daldrup-Link et al, 2003;Matus-zewski et al, 2005). SPIOs, with a hydrodynamicdiameter of 50 to 180 nm, are more efficientlyphagocytosed than USPIOs with sizes of 20 to50 nm. The maximum intracellular iron oxide con-centration ofin vitro-labeled, isolated human macro-phages is 50 pg Fe/cell for the SPIO Ferucarbotran,whereas for USPIO (SHU 555 C), it is < 8 pg Fe/cell(Metzet al, 2004). Besides the particle size of the ironoxides, phagocytic uptake is also dependent onnanoparticle surface properties (i.e., neutral versuscharged). The relative surface properties may have agreater impact on the effectiveness of phagocytosisthan particle size. For example, the ionic Ferucarbo-tran, with a mean diameter of 62 nm, has asignificantly higher cellular uptake compared withnonionic ferumoxides, with a mean diameter of150 nm (Metz et al , 2004). Complex formationbetween ferumoxides and a variety of transfectionagents occurs through electrostatic interactions andenhances the iron uptake in multiple cell types.

Arbab et al showed that the low-molecular weightcationic peptide, protamine sulfate, enhances theuptake of ferumoxides in vitro (Arbab et al, 2004a;Arbab et al, 2004b). Protamine sulfate also doubledthe in vivo uptake of ferumoxides in rats (Wuet al,2007). In our groups study, in vivo peripheralmononuclear cells showed minimal uptake offerumoxtran-10 or ferumoxytol even in the pre-sence of IV protamine sulfate (Wu et al , 2007).However, other groups have shown enhanceduptake of USPIOs by activated monocytes in vitro(Metz et al, 2004).

With respect to contrast-enhancement imagingproperties of CNS tumors, besides their vascularity,

the number and the distribution of activated inflam-matory cells seems to be the most relevant. A largenumber of macrophages and activated microglia havebeen reported to be present within and aroundmalignant brain tumors. The microglial infiltrationis typically most prominent at the periphery of alesion and in the surrounding brain tissue. Theability to track USPIO-labeled cells using MRI andthen correlate them with histology is an importantnew technology in the investigation of many CNSpathologies. Our group has tracked ferumoxtran-10from brain into the cervical lymph nodes of rats(Muldoonet al, 2004). This connection has not been




9/21

well characterized in humans, but may be implicated

in the pathogenesis of diseases such as multiplesclerosis (MS) and Alzheimers disease throughperipheral immune responses to CNS proteins.

Fleige et alhave shown that microglia can readilybe labeled with magnetic particles, and labeled,activated microglia very precisely represents tumormorphology. They found that labeled microglia canbe detected with MRI in vivo and then visualizedhistologically using specific stains for iron (e.g.,Perls stain) (Fleige et al , 2001). Other groupshave similarly found that labeling cells in culturewith USPIOs, combined with MR imaging, providesa noninvasive method for serially tracking and

quantifying the fate of transplanted cells in vivo

(Wuet al, 2008).In another study, fragments of human malignant

glioma were orthotopically xenografted into thebrains of nude mice. All mice underwent MRIexamination 24 h after IV administration of ferumox-tran-10. In this study, Kremer et alobserved a strongcorrelation between tumor-to-background contrastand proliferative index, between tumor-to-back-ground contrast and tumor growth, and betweentumor-to-background contrast and Perls stainingscore, indicating the potential for improved diag-nostic specificity with respect to tumor progression(Kremer et al, 2007).

Figure 3 (A) Cartoon depiction of cerebral blood volume (CBV) and mean transit time (MTT) calculation using DSC first-pass MRI.The first-pass effect of a contrast agent bolus in brain tissue is monitored using a series of T2*-weighted MR images from the samebrain region, which is scanned repeatedly. The susceptibility effect of a paramagnetic (GBCA), or superparamagnetic (USPIO),contrast agent causes decreased signal intensity that can be converted into a contrast agent concentration. This concentration canthen be converted into quantitative measurements of CBV proportional to the area under the curve (hatched area) assuming that the

longitudinal relaxation time T1of bulk tissue remains constant during bolus passage. This assumption holds true if the contrast agentis confined to the vascular space and the vascular volume fraction is so small that its contribution to the overall signal intensity maybe neglected. (B) Comparison of gadodiamide versus ferumoxytol perfusion imaging in a highly vascular and permeable humanglioma (U87MG cell line) xenograft at 12 T. The curves depicted (B.1 and B.4) show the signal intensitytime course usingstandard rapid T2*-weighted gradient echo MR sequences during gadodiamide (B.1) and ferumoxytol (B.4) first-pass boli. Regions ofinterest were defined on the T2-weighted anatomical images (B.2 and B.5) in the tumor (red) and normal-appearing brain tissue(blue). Because of extravasation of the smaller molecular weight gadodiamide from the vasculature, CBV calculations (area under thecurve proportional to area of DSC dip) will be inappropriately low. The CBV parametric maps (B.3 and B.6) show the discrepancybetween CBV calculations using these two agents; ferumoxytol-based perfusion imaging correctly delineates elevated CBV in thisaggressive tumor model. Adapted with permission fromNeuwelt et al (2007)and Varallyay et al (2009).


2



10/21

Monitoring Antiangiogenic Therapy in CentralNervous System Tumors with UltrasmallSuperparamagnetic Iron Oxide Particles

The blockade of neoangiogenic signaling pathways isone of the several key strategies in the treatment ofhigh-grade malignant brain tumors. Bevacizumab,

which neutralizes the vascular endothelial growthfactor-A, is the most commonly used monoclonalantibody for the treatment of high-grade gliomas inhumans (Claes et al, 2008). Growing evidence sug-gests that USPIO-enhanced MRI will be useful in theevaluation of tumor response to antiangiogenictherapy.

Claes et al compared conventional T1-weightedGBCA-enhanced MRI with T2*-weighted USPIO(ferumoxtran-10)-enhanced MRI in mice carryingorthotopic U87 glioma treated with or without theantiangiogenic compound vandetanib. In untreatedanimals, vessel leakage within the tumor, and arelatively high tumor blood volume resulted in good

MRI visibility with GBCA- and USPIO-enhancedMRI. Vandetanib treatment restored the integrity ofthe BTB, resulting in loss of tumor detectability withGBCA, but not with USPIO-enhanced MRI, mostlikely because of continued infiltration of USPIO-labeled macrophages (Claeset al, 2008). In a separatepreclinical study, Robinson et al investigated theutility of susceptibility contrast MRI with a USPIO(feruglose) to assess rat GH3 prolactinomasbefore and 24 h after treatment with either saline or50mg/kg ZD6126. ZD6126 (N-acetylcolchinol-O-phosphate) is a water-soluble phosphate pro-drugof the tubule-binding agent N-acetylcolchinol. It

inhibits tubulin polymerization that disrupts thecytoskeleton of proliferating tumor endothelial cellsthereby leading to endothelial cell detachment,tumor vessel occlusion, and central hemorrhagicnecrosis. Irrespective of treatment, tumor volumesignificantly increased over 24 h. However, saline-treated tumors showed no statistically significantchange in tumor fractional blood volume, whereas asignificant 70% reduction in tumor fractional bloodvolume was observed in the ZD6126-treated cohort.Uptake of the nuclear stain Hoechst 33342, indica-tive of viable tumor cells, was significantly reducedand restricted to the rim of the ZD6126-treatedtumors. A significant positive correlation between

posttreatment tumor fractional blood volume andHoechst 33342 uptake was also observed, providingvalidation of the MRI-derived measurements offractional tumor blood volume as a method oftracking therapeutic response to antiangiogenicagents (Robinsonet al, 2007).

Ongoing clinical trials conducted by our groupshow that USPIOs, combined with PWI, are animportant adjuvant for monitoring tumor responseand for discrimination of pseudo-progression fromtrue tumor progression. It is generally accepted thatincreased malignancy is associated with increasedvascularity and tumor growth is correlated with

neoangiogenesis. In CNS malignancies, CBV has beenone of the most commonly used parameters toestimate microvascular density (Cha et al , 2002).However, in tumors with a disrupted BTB (such asmalignant glioma) leakage of GBCAs from tumorvessels causes inaccurate estimation of tumor CBVusing PWI (Figure 3B) (Neuwelt et al, 2007;Uematsu

and Maeda, 2006). Although the clinical impact ofthis inaccuracy is not known, it could be significant incases in which PWI is used to monitor therapeuticresponse to antiangiogenic chemotherapies, such asbevacizumab. In this scenario, the use of a blood-poolagent, such as the bolus-injectable USPIO ferumox-ytol, would be favorable by eliminating the perme-ability dependence of CBV estimation (Figure 3B).

The evidence regarding CBV normalization andsurvival of patients with brain tumors is notstraightforward, however. One clinical study invol-ving 19 patients with grades 2, 3, and 4 gliomasrevealed a stronger correlation of survival withnormalized CBV than with intensity of GBCAenhancement (Lev et al , 2004). Another studyinvestigating 28 patients with GBM found nosignificant relationship between median CBV andsurvival (Oh et al , 2004). However, one mustconsider that median CBV ignores the intrinsicheterogeneity of tumor perfusion (Aronen et al ,1994;Donahueet al, 2000;Knoppet al, 1999;Kremeret al, 2002;Lev et al, 2004;Sugahara et al, 1998). In23 patients with high-grade gliomas, the fractionaltumor volume of high CBV, which accounts for theheterogeneity of CBV, was analyzed before radio-therapy and it predicted survival. Subsequentchanges in CBV during conformal radiotherapy were

also predictive (Caoet al, 2006). More accurate CBVmaps, generated using USPIOs, may also be useful inthe evaluation of treatment response and helpful inthe differentiation of recurrent tumor from postsur-gical/postirradiation changes.

It is hypothesized that CBV measurements mayprovide a mechanism to distinguish pseudoprogres-sion versus true progression of malignant gliomasafter chemoradiotherapy using temozolomide. Up to50% of patients treated with temozolomide haveincreased areas of GBCA enhancement in the first 3months after completion of radiation (Brandsmaet al , 2008). The PWI using blood-pool agents(USPIOs, such as ferumoxytol) may allow differen-

tiation of true tumor progression from pseudopro-gression based on increased versus decreased CBV,respectively (Figure 4) (unpublished data).

Imaging Stroke with Ultrasmall SuperparamagneticIron Oxide Particles

Imaging ischemic CNS lesions is time dependent.In the past, parenchymal ischemic injury could onlybe detected 6 to 12 h after the onset of symptomsusing standard MRI sequences (Yuh et al, 1991).Diffusion- and perfusion-weighted MRI, using




11/21

USPIOs or GBCAs, allows for the identificationof ischemic lesions earlier and may permit themonitoring of the effects of therapeutic strategies(Chenevert et al, 1991; Moseley et al, 1990; Rosenet al , 1990; Stroke, 1989. Recommendations on

stroke prevention, diagnosis, and therapy. Reportof the WHO Task Force on Stroke and OtherCerebrovascular Disorders, 1989). Contrast agentsthat cause a regional signal loss because of magneticsusceptibility-induced T2* shortening (e.g., USPIOs)

Figure 4 (A) Discordance of rCBV calculated using ferumoxytol versus GBCA enhancement suggestive of pseudoprogression. At themiddle of radiation time point (middle row), rCBV with ferumoxytol clearly shows increased blood flow (yellow/green area on rCBVwith Fe parametric map marked with small red arrow) in a second posterior frontal lobe lesion (this lesion was not initially radiatedafter the patients first resection of a left frontal GBM). After completion of radiation (bottom row), the area of GBCA enhancement isincreased; however, DSC-MRI with ferumoxytol (third column) shows decreased rCBV compared with surrounding normal brain. ( B)A 19-year-old man with GBM imaged pre- and postoperatively, after standard radiochemotherapy (RCT) and at multiple time pointsafter treatment with bevacizumab. B.1: GBCA-enhanced, T1-weighted MRI before surgery shows a large ring enhancing lesion in theright parietooccipital lobe with mass effect and midline shift; B.2: postoperative image, before initiating RCT. B.3: MRI 1 month aftercompletion of RCT revealed increased area of GBCA enhancement on T1-weighted images (see white arrow) with predominantly lowblood volume except for a thin area of high blood volume at the periphery of the Gd- and Fe-rCBV parametric maps (B.4 and B.5).

Note that the peripheral rCBV is more prominent (intense green/yellow circle marked by white arrow in image 5; the red area inimage 4 is likely artifact) using ferumoxytol versus gadoteridol. Adjuvant temozolomide treatment was continued and bevacizumabtreatment was then initiated. B.67: GBCA-enhanced, T1-weighted MRIsafter first and second courses of treatment withbevacizumab show significant improvements in mass effect and diminished contrast enhancement. B.810: after six treatments withbevacizumab and temozolomide, there is a slight increase in GBCA enhancement, but decreased rCBV on both gadoteridol andferumoxytol perfusion imaging.


2



12/21

have been shown to provide substantial contrastbetween ischemic and normally perfused brainareas (Gore and Majumdar, 1990; Villringer et al,1988). The MTT abnormalities, taken togetherwith areas of restricted diffusion on diffusion-weighted imaging, are the preferred techniques fordetermining regions of infarction versus penumbra

(Schaefer et al, 2008).In addition to ischemic changes, according to

experimental data, brain inflammation is presentduring the acute stage of an ischemic stroke, whichmay be optimally imaged using MRI in combinationwith USPIOs (del Zoppoet al, 2000;Stollet al, 1998).There is a growing appreciation for the dual role ofinflammation in stroke, with microglia implicatedearly and macrophages (both bone-marrow derivedand brain parenchymal) implicated later in theestablishment of a chronic, deleterious, proinflam-matory state. Audebert et alshowed that an increasein systemic inflammatory parameters correlates sig-nificantly with lesion volume and stroke severity(Audebert et al, 2004; Nighoghossian et al, 2007).The microglia, activated within minutes of ischemiconset, are involved not only in tissue damage, butultimately may protect the brain against furtherischemic injury (Jander et al, 2007; Schilling et al,2003; Schroeter et al, 1997; Tanaka et al, 2003).Denes et al showed that in mice, after transientmiddle cerebral artery occlusion, microglial prolif-eration was the main factor behind the increasednumber of mononuclear phagocytes seen during thefirst 3 days; the number of activated microglial cellsnegatively correlated with the extent of ischemicbrain damage (Denes et al, 2007).

Work in other experimental stroke models showedthat diffusion- and perfusion-weighted MRI usingGBCAs is not able to differentiate inflamed fromnoninflamed infarct subareas (Schroeter et al, 2001).However, using USPIOs and T2*-weighted imaging,hypointense areas indicative of USPIO-laden inflam-matory cells can be visualized. These areas ofhypointensity were then confirmed by histologicand electron microscopic analyses to correspondwith brain sections infiltrated with USPIO-ladenmacrophages (Rauschet al, 2001;Salehet al, 2004b).

Several experimental and clinical investigationshave been performed to identify the optimal time-window for detecting inflammation after ischemic

injury. Experimental data reveal reproducible US-PIO-induced enhancement as early as 24 h afterischemia. In animal studies, ED1 + cells, a cellmarker for macrophages, were found around the coreof the infarct within the first 24 h after occlusion ofthe middle cerebral artery. On days 2 to 4, USPIOswere found mainly between the core and theperiphery of the lesions and, by day 7, they wereseen only at low concentration both within andaround the lesion (Rausch et al , 2001). Thesefindings may be important in the design andmonitoring of future neuroprotective trials, espe-cially those involving antiinflammatory agents.

In a single-center, open-labeled, clinical phase IIstudy, the potential for USPIO-enhanced MRI versusconventional GBCA-enhanced MRI to image macro-phages in human ischemic stroke lesions was tested.Ten consecutive patients received IV ferumoxtran-10at the end of the first week after symptom onset.Two follow-up MRI scans were performed 24 to 36 h

and 48 to 72 h after infusion. The USPIO-inducedsignal alterations in the ischemic area were evidenton both T1-weighted and T2/T2*-weighted imaging.Contrast enhancement was observed primarily at theperiphery of the infarcted parenchyma. Digitalsubtraction of GBCA-enhanced regions revealeddistinct areas of USPIO enhancement, indicatingthat these areas of enhancement were not because ofBBB disruption, but rather a consequence of iron-labeled macrophage infiltration (Figure 5A) (Salehetal, 2004a; Saleh et al, 2007). There are very limiteddata on the utility of USPIOs in stroke. However,Henning et al recently completed an excellentpreclinical study, which provides further proof-of-principle that this type of imaging may be useful inthe development of future neuroprotective strategies(Henning et al, 2009).

Imaging Carotid Atherosclerosis with UltrasmallSuperparamagnetic Iron Oxide Particles

Carotid artery atherosclerosis can be determined byMR angiography using GBCA or USPIO (especially inpatients with renal failure), which also helps toverify the severity of stenosis (Figure 5B). However,recently it has been recognized that the composition

and stage of atherosclerotic carotid plaques, ratherthan the severity of stenosis they induce, areimportant properties for assessing stroke risk. Inexperimentally induced hyperlipidemic rabbits, US-PIOs of a diameter similar to that of low-densitylipoprotein, 15 to 25 nm, enter and accumulate inatherosclerotic plaques with high macrophage con-tent (Ruehmet al, 2002;Tanget al, 2006). Cappendijket al studied 11 patients with recurrent transientischemic attacks and ultrasound-proven carotidstenosis scheduled for carotid endarterectomy usingferumoxtran-10 (Cappendijk et al, 2008). Significant,detectable signal changes were found onin vivoT2*-weighted gradient echo MR images acquired 24 h

after IV administration of USPIO. After surgery,immunohistochemistry of plaque samples showedferumoxtran-10 accumulation predominantly withinmacrophages in ruptured and rupture-prone athero-sclerotic lesions. In an additional study, 20 patientswith symptomatic carotid stenosis, all scheduled forcarotid endarterectomy, were imaged before and 36 hafter ferumoxtran-10 infusion (Tang et al , 2006).Symptomatic carotid plaques showed significantlymore inflammatory activity than did the contralateralcarotid artery. Despite a mean contralateral carotidstenosis of only 46%, 95% of these asymptomaticplaques showed USPIO uptake, suggesting an




13/21

inflammatory burden within the carotid atheromasbilaterally. This finding correlates with the propen-sity for patients with symptomatic carotid stenosisto have bilateral disease. As the majority (80%) ofembolic infarcts related to carotid stenosis presentwithout any warning, USPIO-enhanced detection ofinflammation within plaques may serve as animportant screening tool to reduce the incidence ofstroke. It may also be useful for tracking treatmentresponse to lipid-clearing medications. Other novel

techniques for studying atherosclerotic carotid pla-ques include very interesting work by Strijkersgroup using GBCA-loaded lipososomes in combina-tion with MRI for detection of intimal thickening(Mulder et al, 2006).

Imaging Autoimmune Disorders with UltrasmallSuperparamagnetic Iron Oxide Particles

Multiple Sclerosis and acute disseminated encepha-lomyelitis (ADEM) are immune-mediated disordersof the CNS. Several observations suggest a major role

of macrophages in axonal injury, but the triggers forthis autoimmune response have yet to be discovered(Bitschet al, 2000;Brochetet al, 2006). Experimentalautoimmune encephalomyelitis (EAE) is an animalmodel of human MS. It can be induced in severalspecies by administration of myelin antigens ormyelin-reactive CD4 + cells (Rausch et al, 2003). InEAE, USPIO-enhanced MRI reveals areas of hypoin-tensity on T2*-weighted images, which correspond toUSPIO-laden mononuclear cells within inflamma-

tory lesions. Immunohistochemical analysis alsoshows that the iron particles are specifically loca-lized within newly infiltrated ED1 + cells, but not inED2 + perivascular macrophages (Floris et al, 2004).

In both animal and human studies of MS andADEM, USPIO-enhancement patterns differ fromGBCA enhancement in time (Figure 6) (Bendszuset al, 2005; Dousset et al, 2006; Floris et al, 2004;Rauschet al, 2003;Rauschet al, 2004;Vellingaet al,2008). Rausch et alfound that this discrepancy wasmost prominent during the first relapse, in which alarge number of USPIO-enhancing areas did notshow any GBCA uptake (Rausch et al, 2003;Rausch

Figure 5 (A) Reprinted with permission from Saleh et al (2007). The USPIO-enhanced MRI of a 54-year-old man with infarctioninvolving the left middle cerebral artery distribution reveals the differential development of USPIO-related signal changes over time.Compared with the nonenhanced, T1-weighted image 5 days after stroke (A.1), T1-weighted images 24 h (A.2), and 48 h (A.3) afterUSPIO infusion show increasing hyperintense signal enhancement in the periphery of the infarcted parenchyma. Nonenhanced, T 2*-weighted image (A.4) displays hyperintense demarcation of the infarcted territory. T2*-weighted images 24 h (A.5) and 48 h (A.6)after USPIO infusion show signal change from hyperintense to hypointense attributable to USPIO perfusion. (B) Ferumoxytol MRangiography at 3 T of the supraaortic arteries in a kidney transplant patient with a glomerular filtration rate (GFR) < 60 mL/mins/1.73 m2. Large plaque causes significant luminal narrowing at the origin of the innominate artery (red arrows). A significant stenosiswas also observed at the origin of the left subclavian artery (not shown). Carotid bulb shows moderate stenosis on both sides (white

arrows). Note the difference in diameter between the left and right carotid as well as the vertebral arteries. The color reproduction ofthis figure is available on the html full text version of the manuscript.


2



14/21

et al, 2004). Intracellularly located USPIO particleswere concluded to be responsible for enhancementin 19 patients with MS, even in areas of intact BBB(Vellinga et al , 2008). One might conclude thatmacrophages enter the brain during acute flares ofMS independently from breakdown of the BBB asdefined by GBCA enhancement (Manninger et al,2005).

Baeten et al showed that the timing of USPIOinjection and imaging determines the amount anddistribution of inflammatory lesions visualized. Atdisease onset, USPIOs detected lesions in the caudalpart of the brainstem, whereas at the peak of plaqueseverity, USPIOs mainly target inflammatory regionsin the midbrain with only small amounts in the

caudal part of the brainstem (Baeten et al, 2008). Inrats with EAE, neurologic deficits correlate with theMRI results.

Miki et al found a negative correlation betweenGBCA-enhancing lesion volume and duration ofdisease, suggesting that the BBB abnormalities areless important over time (Miki et al, 1999). Further-more, when the disease evolves to the secondary-progressive stage, decreasing levels of enhancementare observed despite increasing neurologic deficits,again suggesting a diminished role of the BBBabnormality revealed by GBCAs (Mikiet al, 1997).

Acute axonal injury and irreversible axonal lossare generally accepted to be major factors in the

pathophysiology of MS. Brochet et al studied therelationship with inflammation and demyelinationin Dark Agouti rats with severe protracted-relapsingEAE. This model, characterized by an initial attackwith very early axonal damage, shows the conse-quence of early inflammation and demyelination.The second attack is characterized by both macro-phage infiltration of the CNS, axonal loss, andincreased areas of demyelination. Compared withacute models of EAE, where all animals present withMRI signal changes in the CNS at clinical diseaseonset, relapsing EAE models show MRI signalalterations after USPIO injection, which are not

constant (Brochet et al, 2006). To validate the useof USPIOs as a noninvasive tool for evaluatingtherapeutic strategies in EAE, Floris et al treatedEAE animals with the immunomodulator, 3-hydro-xy-3-methylglutaryl Coenzyme A reductase inhibitoror lovastatin; MRI revealed that the USPIO load inthe brain was significantly diminished in lovastatin-treated animals and this correlated with improvedclinical scores (Floris et al, 2004).

Improvements in early disease detection areclearly needed in this field; an exciting alternativeto USPIO-enhanced MRI is work being conducted bySibson et al, using antibody-conjugated iron oxidemicroparticles. Antibodies to vascular cell adhesionmolecule-1 conjugated to an MRI contrast agent may

allow for earlier detection, estimates of diseaseseverity, and monitoring of therapy (McAteer et al,2007).

Imaging Central Nervous System Trauma withUltrasmall Superparamagnetic Iron Oxide Particles

The development of neuroregenerative therapies forpatients with traumatic brain or spinal cord injuriesis no longer science fiction. Embryonic stem cellsand other progenitor cell populations, combinedwith biocompatible structural matrices, are beinginvestigated for their potential to restore function

after trauma (Sykova and Jendelova, 2007). Criticalfactors for the successful application of theseregenerative cell therapies include, the ability oftransplanted cells to migrate from the site oftransplantation to the lesioned area; to survive,differentiate, and/or produce growth factors andcytokines for the prolonged periods of time necessaryfor the patient to benefit from their regenerativeproperties (Sykova and Jendelova, 2007). Implanta-tion of stem cells may improve functional recoveryafter experimental models of brain and spinalcord injury (Bareyre, 2008; Kulbatski et al, 2005;McDonald et al, 1999;Sykova and Jendelova, 2007).

1 2 3

Coronal Coronal

4

Figure 6 Adapted with permission from Manninger et al (2005). Patient with ADEM. Axial, T1-weighted images without (1), andwith (2) gadolinium (inset, coronal) show faint, subtle enhancement in multiple brain stem lesions. Six days later, significant andmore prominent enhancement can be seen at the same sites (3) using ferumoxtran-10 (inset, coronal). Three months later, thelesions no longer enhance on T1-weighted images with gadolinium (4).




15/21

The USPIOs may serve as useful adjuncts fornoninvasive anatomic and temporal tracking of stemcells in CNS trauma and stroke (Hoehn et al, 2007;Weberet al, 2006).

Although there are limitations to USPIO-basedmolecular imaging, preclinical studies may benefitfrom the combination of magnetic cell labeling andtracking in vivowith MRI. An IV delivery of USPIO-labeled embryonic stem cells into rats injured withphotochemical cortical lesions led to MR-visiblemigration of labeled cells into the lesion. Histology

confirmed that 70% differentiated into astrocytes,< 1% oligodendrocytes, and B5% became neurons(Sykova and Jendelova, 2007). This same group alsoinvestigated balloon-induced spinal cord injurytreated with ferumoxide-labeled nonhematopoieticmesenchymal stem cells. The labeled cells could betracked with MRI and led to improved locomotor andplantar test results compared with control animals(Sykova and Jendelova, 2007).

In a controlled cortical injury model of TBI in rat,pooled leukocytes labeled with ferumoxides can bedetected histologically around the periphery of thelesion. In the setting of experimental TBI, MRI

tracking of USPIO-labeled inflammatory cells isdifficult acutely using T2*-weighted imaging se-quences because of the presence of free red bloodcells. However, pre- and postferumoxytol-enhancedimages may be useful for quantifying inflammation(Figure 7) (unpublished data). Dynamic imagingusing ferumoxytol for the evaluation of cerebralperfusion and BBB permeability in the acute andchronic stages after TBI, may improve our under-standing of the effects of TBI on cerebral hemody-namics. Information about edema formation, and

monitoring therapeutic responses to treatments de-signed to reduce edema and inflammation, will beimportant in the translation of these novel strategiesto humans.

Imaging Epilepsy with Ultrasmall SuperparamagneticIron Oxide Particles

Akhtari et al used a rat model of temporal lobeepilepsy and a-methyl tryptophan (AMT)-taggeddextran-coated USPIOs to evaluate the potential forfunctional MRI localization of epileptogenic foci

Figure 7 (A) Pre- and postferumoxtyol (15 mins, middle row; 24 h, bottom row) enhanced 12 T MRIs after controlled cortical injuryin a rat model of traumatic brain injury. At 72h after injury, there is marked signal loss visualized on T2* (Fe 24 h images);(B) Phagocytic inflammatory cells, seen on H&E (B.1) and stained with CD68 (B.2) and fibronectin (B.3). ( C) The inflammatory cellsare at least in part derived from peripheral circulating monocytes as illustrated in panels (C.1) and (C.2), which show quantum dot(Invitrogen, Carlsbad, CA)-labeled peripheral mononuclear cells infiltrating around the cortical injury (#1; 800 magnification) orwithin blood vessels on the uninjured side (C.2; 800 magnification).


2



16/21

(Akhtari et al, 2008). AMT is preferentially taken upin epileptogenic foci and reflects increased serotoninproduction or induction of the kynurenine pathway(Juhasz et al, 2004). In acute studies, AMT USPIOsinjected 3 days after kainic acid-induced statusepilepticus localized to bilateral hippocampi,whereas plain USPIOs were only observed unilateral

to the lesion, presumably because of inflammation(Akhtari et al, 2008). In chronic studies, the authorscorrelated AMT USPIO uptake with the occurrenceof spontaneous seizures; MRI-localization of theUSPIOs agreed with electroencephalography. Ad-vantages of this technique, compared with currentdiagnostic algorithms, are multiple: no radiationexposure as with single photon emission computedtomography allows for repeat imaging; uses standardMRI; the tracer is easy to synthesize in a short timeframe and is inexpensive. Although USPIOs haveshown no evidence of CNS toxicity as discussedabove, the long-term fate of these agents in the brainneeds to be determined, as free iron is known to beepileptogenic (Willmoreet al, 1978).

Emerging Therapeutic Applications Using UltrasmallSuperparamagnetic Iron Oxide Particles

The therapeutic applications of USPIOs are alsorapidly expanding as the chemistry and throughputfor production is streamlined. These agents can beconjugated to drugs, proteins, enzymes, antibodies,or nucleotides and can be directed to an organ,tissue, or tumor using an external magnetic field(Laurent et al, 2008). These same surface modifica-

tions, used for diagnostic specificity, will enhancetargeting for drug-delivery, gene therapy, radiosensi-tization, radiation therapy planning with MRI, tissuerepair, detoxification, and magnetic fluid hyperther-mia (Chertok et al, 2008;Hartman et al, 2008;Khooand Joon, 2006;Maier-Hauffet al, 2007;Thoreket al,2006;van Landeghem et al, 2009).

The USPIOs can be used for hyperthermic ablationof CNS tumors after direct inoculation into tumors,or IV administration, depending on the agentsbiodistribution. Once targeted, particles are exposedto an alternating magnetic field, which produceselectrical current and subsequent energy dispersionin the form of heat. Superparamagnetic species with

single magnetic domains, dissipate heat as a result ofrelaxation of the domain dipole, a process known asNeel relaxation (Hartman et al, 2008). It takes farlower-strength magnetic fields, using VSPIOs (3 to7 nm), to achieve the same level of heating as largerferromagnetic agents. One limitation of this treat-ment strategy has been target specificity. However, afeasibility trial was recently conducted using ami-nosilane-coated iron oxide nanoparticles in patientswith recurrent glioblastoma (Maier-Hauffet al, 2007).In this study, Maier-Hauff et al inoculated 14patients, using multiple stereotactic-guided injec-tions. Multiple sessions of thermotherapy were well

tolerated; the median maximum temperature was44.61C (431C is generally felt to be necessary fortumor cell ablation) and there were signs of localtumor control. A phase II trial is currently underway.Additionally, postmortem studies of patients en-rolled in the feasibility trial showed that dispersedparticles and particle aggregates were phagocytosed

mainly by macrophages, whereas glioblastoma cellsshowed uptake to a minor extent. Van Landeghem etaldid not observe deleterious bystander effects ofmagnetic fluid hyperthermia such as sarcomatoustumors or sterile abscess formation, nor did theyobserve any foreign body giant cell reaction (vanLandeghem et al, 2009).

Risk and Utility of Ultrasmall Superparamagnetic IronOxide Particles in Comparison to Gadolinium-basedContrast Agents

Although there is solid evidence of the deleterious

effects of nanoparticles and in some cases iron oxidenanoparticles, SPIOs appear quite safe and USPIOs,in particular ferumoxytol, appear to be remarkablysafe, as evidenced by the recent FDA approval. TheFDA-approved package insert for ferumoxytol (Fer-aheme) reviews three randomized clinical trials witha total of 1726 patients of whom 1562 had chronickidney disease and were thereby chronically illpatients. Despite this, only two patients had sig-nificant reactions to ferumoxytol and these reactionswere transient. It appears that in patients who do notsuffer from iron overload, ferumoxytol even inseriously ill patients is quite safe.

With regard to its use in comparison to GBCAs, aswe have tried to show in this review, except inpatients with compromised kidney function, USPIOsare a supplement, rather than a replacement forGBCAs as contrast agents. In a head-to-head compar-ison, GBCAs are clearly a better screening agent, inpart because of the rapid leak into CNS pathologiclesions. With ferumoxytol, it takes 24 h after contrastadministration to get analogous images. In addition,in dynamic imaging it appears that Gd is superior toassess permeability (dynamic contrast enhance-ment), whereas when assessing blood volume andflow, USPIOs such as ferumoxytol act as a blood-poolagent at early time points, and thereby may give more

accurate measurements of blood volume and bloodflow. As USPIOs specifically target phagocytic cells,that is macrophages, they may also be better forassessing inflammation.

Conclusions

Superparamagnetic iron oxide nanoparticles are animportant development for investigation and poten-tial therapeutics across a wide variety of systemicand CNS pathologies. Although there is no perfectMRI contrast agent yet, USPIOs satisfy many of the




17/21

requirements that limit paramagnetic gadolinium-chelate contrast agents. The USPIOs appear to besafe, are easy to administer, and provide significantcontrast enhancement, even on low tesla magnets.They may serve as complimentary agents to improvelocalization, characterization, and follow-up in di-verse neurologic lesions. In CNS tumors, USPIOs

may improve detection and diagnosis and, usingdynamic studies, will be invaluable in the future formonitoring therapeutic responses to antiangiogenicchemotherapies and for differentiating true tumorprogression from pseudoprogression. The USPIOsmay also serve as safe alternative contrast agents inpatients with renal dysfunction who are at risk fornephrogenic systemic fibrosis with GBCA-basedcontrast agents. Although USPIOs such as ferumox-ytol are not the answer to all diagnostic andtherapeutic applications currently envisioned, manyof these ideas are years, if not decades, from clinicalpractice. Ferumoxytol, in particular, is an excitingand powerful tool that is FDA approved and can beused to study a variety of CNS pathologies.

Acknowledgements

We thank Ms. Audrey Selzer for her assistance with12T MRI acquisitions, Mr. William Woodward forcollecting data for Figure 5b, and Ms. EmilyHochhalter for her administrative assistance. Thisresearch was funded in part by the NationalInstitutes of Health grants NS33618, NS34608,NS053468, and NS44687 from the National Institute

of Neurological Disorders and Stroke, a Departmentof Defense Center of Excellence Award, AMAGPharmaceuticals Inc, and by the Department ofVeterans Affairs, all to EAN. This research was alsofunded in part by a Neurosurgery Research andEducation Foundation grant sponsored by Codman,a Johnson & Johnson Company to JSW, NIH RO1-EB007258 to WDR, The Oregon Opportunity and agrant from the WM Keck Foundation.

Disclosure/conflict of interest

The authors declare no conflict of interest.

References

Akeson P, Nordstrom CH, Holtas S (1997) Time-depen-dency in brain lesion enhancement with gadodiamideinjection.Acta Radiol38:1924

Akhtari M, Bragin A, Cohen M, Moats R, Brenker F, LynchMD, Vinters HV, Engel Jr J (2008) Functionalizedmagnetonanoparticles for MRI diagnosis and localiza-tion in epilepsy.Epilepsia49:141930

Arbab AS, Yocum GT, Kalish H, Jordan EK, Anderson SA,Khakoo AY, Read EJ, Frank JA (2004a) Efficientmagnetic cell labeling with protamine sulfate com-

plexed to ferumoxides for cellular MRI. Blood104:121723

Arbab AS, Yocum GT, Wilson LB, Parwana A, Jordan EK,Kalish H, Frank JA (2004b) Comparison of transfectionagents in forming complexes with ferumoxides, celllabeling efficiency, and cellular viability. Mol Imaging3:2432

Aronen HJ, Gazit IE, Louis DN, Buchbinder BR, Pardo FS,

Weisskoff RM, Harsh GR, Cosgrove GR, Halpern EF,Hochberg FH et al. (1994) Cerebral blood volume mapsof gliomas: comparison with tumor grade and histologicfindings.Radiology191:4151

Audebert HJ, Rott MM, Eck T, Haberl RL (2004) Systemicinflammatory response depends on initial stroke sever-ity but is attenuated by successful thrombolysis. Stroke35:212833

Baeten K, Hendriks JJ, Hellings N, Theunissen E, Vander-locht J, Ryck LD, Gelan J, Stinissen P, Adriaensens P(2008) Visualisation of the kinetics of macrophageinfiltration during experimental autoimmune encepha-lomyelitis by magnetic resonance imaging. J Neuroim-munol195:16

Banks WA (1999) Physiology and pathology of the blood-

brain barrier: implications for microbial pathogenesis,drug delivery and neurodegenerative disorders. J Neu-rovirol5:53855

Bareyre FM (2008) Neuronal repair and replacement inspinal cord injury. J Neurol Sci265:6372

Barnett PA, Roman-Goldstein S, Ramsey F, McCormick CI,Sexton G, Szumowski J, Neuwelt EA (1995) Differentialpermeability and quantitative MR imaging of a humanlung carcinoma brain xenograft in the nude rat. Am JPathol146:43649

Bendszus M, Bartsch A, Stoll G (2005) Is the disruption ofthe blood-brain barrier a prerequisite for cellularinfiltration in autoimmune encephalitis? Brain128:E25

Bitsch A, Schuchardt J, Bunkowski S, Kuhlmann T, BruckW (2000) Acute axonal injury in multiple sclerosis.Correlation with demyelination and inflammation.Brain123(Pt 6):117483

Blockley NP, Jiang L, Gardener AG, Ludman CN, FrancisST, Gowland PA (2008) Field strength dependence of R1and R2* relaxivities of human whole blood to ProHance,Vasovist, and deoxyhemoglobin. Magn Reson Med60:131320

Bourrinet P, Bengele HH, Bonnemain B, Dencausse A, IdeeJM, Jacobs PM, Lewis JM (2006) Preclinical safety andpharmacokinetic profile of ferumoxtran-10, an ultra-small superparamagnetic iron oxide magnetic resonancecontrast agent.Invest Radiol41:31324

Brandsma D, Stalpers L, Taal W, Sminia P, van den Bent MJ(2008) Clinical features, mechanisms, and managementof pseudoprogression in malignant gliomas. LancetOncol9:45361

Brekke C, Williams SC, Price J, Thorsen F, Modo M (2007)Cellular multiparametric MRI of neural stem celltherapy in a rat glioma model. Neuroimage 37:76982

Brochet B, Deloire MS, Touil T, Anne O, Caille JM, DoussetV, Petry KG (2006) Early macrophage MRI of inflamma-tory lesions predicts lesion severity and disease devel-opment in relapsing EAE. Neuroimage 32:26674

Cameron DG, Bensley EH, Wood P, Grayston V (1951)Treatment of iron deficiency anaemia with saccharatediron oxide given by the intravenous route. Can MedAssoc J64:2730

Cao Y, Tsien CI, Nagesh V, Junck L, Ten Haken R, Ross BD,Chenevert TL, Lawrence TS (2006) Survival prediction


3



18/21

in high-grade gliomas by MRI perfusion before andduring early stage of RT [corrected]. Int J Radiat OncolBiol Phys 64:87685

Cappendijk VC, Kessels AG, Heeneman S, Cleutjens KB,Schurink GW, Welten RJ, Mess WH, van Suylen RJ,Leiner T, Daemen MJ, van Engelshoven JM, Kooi ME(2008) Comparison of lipid-rich necrotic core size insymptomatic and asymptomatic carotid atherosclerotic

plaque: initial results. J Magn Reson Imaging 27:135661

Cha S, Knopp EA, Johnson G, Wetzel SG, Litt AW, Zagzag D(2002) Intracranial mass lesions: dynamic contrast-enhanced susceptibility-weighted echo-planar perfu-sion MR imaging. Radiology223:1129

Chang KH, Ra DG, Han MH, Cha SH, Kim HD, Han MC(1994) Contrast enhancement of brain tumors atdifferent MR field strengths: comparison of 0.5 T and2.0 T. AJNR Am J Neuroradiol 15:14139; discussion14201413

Chenevert TL, Pipe JG, Williams DM, Brunberg JA (1991)Quantitative measurement of tissue perfusion anddiffusionin vivo. Magn Reson Med17:197212

Chertok B, Moffat BA, David AE, Yu F, Bergemann C, Ross

BD, Yang VC (2008) Iron oxide nanoparticles as a drugdelivery vehicle for MRI monitored magnetic targetingof brain tumors. Biomaterials29:48796

Claes A, Gambarota G, Hamans B, van Tellingen O,Wesseling P, Maass C, Heerschap A, Leenders W(2008) Magnetic resonance imaging-based detection ofglial brain tumors in mice after antiangiogenic treat-ment.Int J Cancer122:19816

Corot C, Petry KG, Trivedi R, Saleh A, Jonkmanns C, Le BasJF, Blezer E, Rausch M, Brochet B, Foster-Gareau P,Baleriaux D, Gaillard S, Dousset V (2004) Macrophageimaging in central nervous system and in carotidatherosclerotic plaque using ultrasmall superparamag-netic iron oxide in magnetic resonance imaging. InvestRadiol39:61925

Corot C, Robert P, Idee JM, Port M (2006) Recent advancesin iron oxide nanocrystal technology for medicalimaging.Adv Drug Deliv Rev58:1471504

Daldrup-Link HE, Rudelius M, Oostendorp RA, Settles M,Piontek G, Metz S, Rosenbrock H, Keller U, HeinzmannU, Rummeny EJ, Schlegel J, Link TM (2003) Targeting ofhematopoietic progenitor cells with MR contrast agents.Radiology228:7607

del Zoppo G, Ginis I, Hallenbeck JM, Iadecola C,Wang X, Feuerstein GZ (2000) Inflammation and stroke:putative role for cytokines, adhesion moleculesand iNOS in brain response to ischemia. Brain Pathol10:95112

Denes A, Vidyasagar R, Feng J, Narvainen J, McColl BW,Kauppinen RA, Allan SM (2007) Proliferating residentmicroglia after focal cerebral ischaemia in mice.J CerebBlood Flow Metab 27:194153

Donahue KM, Krouwer HG, Rand SD, Pathak AP, Marszalk-owski CS, Censky SC, Prost RW (2000) Utility ofsimultaneously acquired gradient-echo and spin-echocerebral blood volume and morphology maps in braintumor patients. Magn Reson Med43:84553

Dousset V, Brochet B, Deloire MS, Lagoarde L, Barroso B,Caille JM, Petry KG (2006) MR imaging of relapsingmultiple sclerosis patients using ultra-small-particleiron oxide and compared with gadolinium. AJNR Am JNeuroradiol27:10005

Enochs WS, Harsh G, Hochberg F, Weissleder R (1999)Improved delineation of human brain tumors on MR

images using a long-circulating, superparamagnetic ironoxide agent. J Magn Reson Imaging9:22832

Essig M, Weber MA, von Tengg-Kobligk H, Knopp MV, YuhWT, Giesel FL (2006) Contrast-enhanced magneticresonance imaging of central nervous system tumors:agents, mechanisms, and applications.Top Magn ResonImaging17:89106

Fleige G, Nolte C, Synowitz M, Seeberger F, Kettenmann H,

Zimmer C (2001) Magnetic labeling of activated micro-glia in experimental gliomas. Neoplasia 3:48999

Floris S, Blezer EL, Schreibelt G, Dopp E, van der Pol SM,Schadee-Eestermans IL, Nicolay K, Dijkstra CD, de VriesHE (2004) Blood-brain barrier permeability and mono-cyte infiltration in experimental allergic encephalomye-litis: a quantitative MRI study. Brain127:61627

Gore JC, Majumdar S (1990) Measurement of tissue bloodflow using intravascular relaxation agents and magneticresonance imaging. Magn Reson Med14:2428

Groothuis DR, Vriesendorp FJ, Kupfer B, Warnke PC, LapinGD, Kuruvilla A, Vick NA, Mikhael MA, Patlak CS(1991) Quantitative measurements of capillary transportin human brain tumors by computed tomography. AnnNeurol30:5818

Harpur ES, Worah D, Hals PA, Holtz E, Furuhama K,Nomura H (1993) Preclinical safety assessment andpharmacokinetics of gadodiamide injection, a newmagnetic resonance imaging contrast agent. InvestRadiol28(Suppl 1):S2843

Hartman KB, Wilson LJ, Rosenblum MG (2008) Detectingand treating cancer with nanotechnology. Mol DiagnTher12:114

Henning EC, Ruetzler CA, Gaudinski MR, Hu TC, LatourLL, Hallenbeck JM, Warach S (2009) Feridex preloadingpermits tracking of CNS-resident macrophages aftertransient middle cerebral artery occlusion.J Cereb BloodFlow Metab29:122939

Herschman HR (2003) Molecular imaging: looking atproblems, seeing solutions. Science 302:6058

Hoehn M, Wiedermann D, Justicia C, Ramos-Cabrer P,Kruttwig K, Farr T, Himmelreich U (2007) Cell trackingusing magnetic resonance imaging. J Physiol584:2530

Hunt MA, Bago AG, Neuwelt EA (2005) Single-dosecontrast agent for intraoperative MR imaging of intrinsic

brain tumors by using ferumoxtran-10. AJNR Am JNeuroradiol26:10848

Idiyatullin D, Corum C, Park JY, Garwood M (2006) Fastand quiet MRI using a swept radiofrequency. J MagnReson181:3429

Jander S, Schroeter M, Saleh A (2007) Imaging inflamma-tion in acute brain ischemia.Stroke 38:6425

Juhasz C, Chugani DC, Padhye UN, Muzik O, Shah A,Asano E, Mangner TJ, Chakraborty PK, Sood S, ChuganiHT (2004) Evaluation with alpha-[11C]methyl-L-trypto-phan positron emission tomography for reoperationafter failed epilepsy surgery. Epilepsia45:12430

Ke Y, Ming Qian Z (2003) Iron misregulation in the brain: aprimary cause of neurodegenerative disorders. LancetNeurol2:24653

Khoo VS, Joon DL (2006) New developments in MRI fortarget volume delineation in radiotherapy. Br J Radiol79(Spec No 1):S215

Knopp EA, Cha S, Johnson G, Mazumdar A, Golfinos JG,Zagzag D, Miller DC, Kelly PJ, Kricheff II (1999) Glialneoplasms: dynamic contrast-enhanced T2*-weightedMR imaging. Radiology211:7918

Korosoglou G, Tang L, Kedziorek D, Cosby K, Gilson WD,Vonken EJ, Schar M, Sosnovik D, Kraitchman DL, Weiss




19/21

RG, Weissleder R, Stuber M (2008) Positive contrast MR-lymphography using inversion recovery with ON-reso-nant water suppression (IRON). J Magn Reson Imaging27:117580

Kraemer DF, Fortin D, Doolittle ND, Neuwelt EA (2001)Association of total dose intensity of chemotherapy i

Documents

Superparamagnetic Iron Oxide Nanoparticles