Review Article Superparamagnetic iron oxide nanoparticles: diagnostic magnetic resonance imaging and potential therapeutic applications in neurooncology and central nervous system inflammatory 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 of Neuroradiology, Universitatsklinikum �� Wu ��rzburg, Wu ��rzburg, Germany 4Department of Radiology, Oregon Health and Science University, Portland, Oregon, USA 5Advanced Imaging Research Center, Oregon Health and Science University, Portland, Oregon, USA 6Portland Veterans Affairs Medical Center, Portland, Oregon, USA Superparamagnetic iron oxide nanoparticles have diverse diagnostic and potential therapeutic applications in the central nervous system (CNS). They are useful as magnetic resonance imaging (MRI) contrast agents to evaluate: areas of blood���brain barrier (BBB) dysfunction related to tumors and other neuroinflammatory pathologies, the cerebrovasculature using perfusion-weighted MRI sequences, and in vivo cellular tracking in CNS disease or injury. Novel, targeted, nanoparticle synthesis strategies will allow for a rapidly expanding range of applications in patients with brain tumors, cerebral ischemia or stroke, carotid atherosclerosis, multiple sclerosis, traumatic brain injury, and epilepsy. These strategies may ultimately improve disease detection, therapeutic monitoring, and treatment efficacy especially in the context of antiangiogenic chemotherapy and antiinflammatory medications. The purpose of this review is to outline the current status of superparamagnetic iron oxide nanoparticles in the context of biomedical nanotechnology as they apply to diagnostic MRI and potential therapeutic applications in neurooncology and other CNS inflammatory conditions. Journal of Cerebral Blood Flow & Metabolism (2010) 30, 15���35 doi:10.1038/jcbfm.2009.192 published online 16 September 2009 Keywords: blood���brain barrier CNS tumors magnetic resonance imaging ultrasmall superparamagnetic iron oxide nanoparticles Introduction The diagnosis and treatment of pathologies that affect the central nervous system (CNS) are currently undergoing a renaissance because of the marked proliferation of ���nanoscale��� technologies. Nanotech- nology, as it relates to biomedicine, can broadly be defined as ���nano-sized structures that contain at least one dimension between 1 to 100 nm in sizeyand possess new or enhanced properties that are un- attainable at both smaller (quantum) [and] larger (macromolecular) levels��� (Hartman et al, 2008). Superparamagnetic iron oxide nanoparticles are based on magnetite (Fe3O4), which has received the most 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 utility of superparamagnetic iron oxides as magnetic reso- nance imaging (MRI) contrast agents has been studied for more than two decades (Weissleder et al, 1990) and the list of available agents is rapidly expanding (Table 1). These particles can be Received 3 April 2009 revised 11 August 2009 accepted 13 August 2009 published online 16 September 2009 Correspondence: Dr EA Neuwelt, Department of Neurological Surgery, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, L603, Portland, OR 97239, USA. E-mail: neuwelte@ohsu.edu Journal of Cerebral Blood Flow & Metabolism (2010) 30, 15���35 & 2010 ISCBFM All rights reserved 0271-678X/10 $32.00 www.jcbfm.com

organized according to their hydrodynamic diameter into several categories (Corot et al, 2006): standard superparamagnetic iron oxide particles (SPIOs) (50 to 180 nm), ultrasmall superparamagnetic iron oxide particles (USPIOs) (10 to 50 nm), and very small superparamagnetic iron oxide particles (VSPIOs) ( 10 nm). Most contemporary investigations use USPIOs therefore, for the sake of consistency we will refer to superparamagnetic iron oxide nanopar- ticles, in general, as USPIOs unless specifically discussing SPIOs or VSPIOs. Particles of iron oxide have been administered intravenously (IV) for over 50 years, initially for the treatment of anemia (Cameron et al, 1951). Emerging experimental and clinical applications in the CNS capitalize on both the physical and magnetic proper- ties of iron oxide nanoparticles the list of biomedical imaging applications for these nanoparticles con- tinues to expand. There are a number of important qualities of USPIOs that make them attractive as complimentary or alternative MRI contrast agents compared 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) USPIOs are avidly taken up by phagocytic cells such as the Kupffer cell fraction of the liver, circulating mono- cytes/macrophages and mononuclear T cells, as well as reactive astrocytes, microglia, and dendritic cells within the brain. Neutrophils have not been found to take up USPIO. The USPIOs are cleared from the circulation primarily by the reticuloendothelial system (Bourrinet et al, 2006) these properties (i.e., degree of cellular labeling and rate of clearance) are dependent on size, coating, and method of delivery and will be discussed in detail below. Limitations of these agents for current CNS imaging applications are primarily related to the inability to reliably differentiate USPIO signal from resident brain iron signal (i.e., in the setting of hemorrhage related to stroke or trauma). There are also very little data in humans regarding the long-term clearance of these agents from the brain. Ferumoxides (Endorem, Guerbet in Europe Fer- idex, AMAG Pharmaceuticals Inc in the USA and Japan) and ferucarbotran (Resovist, Schering Bayer in Europe and Japan) are commercially available SPIOs approved for MR imaging of liver tumors (Ros et al, 1995). Clinical CNS imaging studies have also been performed with these compounds (Rose et al, 2006 Varallyay et al, 2002). The USPIO, ferumoxytol (Feraheme, AMAG Pharmaceuticals Inc, Cambridge MA, USA) is approved for iron-replacement therapy in patients with chronic renal failure (Kidney Daily, 6/30/2009). Ferumoxtran-10 (Manninger et al, 2005 Saleh et al, 2007), SHU555C (Vellinga et al, 2008), and ferumoxytol (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 not revealed significant toxicities (Muldoon et al, 2006 Neuwelt et al, 2007). Ferumoxytol, in particular, is attractive as an MRI contrast agent because it can be given as a bolus for first-pass perfusion imaging and appears to be safe in patients with chronic kidney disease at later time points (i.e., 24h), ferumoxytol accumulation is evident in areas of blood���brain barrier (BBB) dysfunction that may be fundamentally related to inflammation from any cause. This review will focus primarily on USPIOs, specifically ferumoxtran-10 and ferumoxytol, for diagnostic applications in the CNS with an emphasis on their utility as MRI contrast agents in the setting of 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 (mM 1 sec 1)b Ferumoxides AMI-25 Feridex/Endorem Guerbet AMAG Pharm. Inc Dextran T10 120���180 (SPIO) 30 r1 =10.1 r2 =120 Ferucarbotran SH U 555 A Resovist Bayer Schering Pharma AG Carboxydextran 60 (SPIO) 8���12 r1 =9.7 r2 =189 Ferumoxtran-10 AMI-227 Combidex/Sinerem Guerbet AMAG Pharm. Inc Dextran T10, T1 15���30 (USPIO) 45 r1 =9.9 r2 =65 Ferumoxytol Code 7228 AMAG Pharm. Inc Polyglucose sorbitol carboxymethyl ether 30 (USPIO) 18���74 r1 =15 r2 =89 SH U 555 C Supravist Bayer Schering Pharma AG Carboxydextran 21 (USPIO) 40 r1 =10.7 r2 =38 Feruglose NC-100150 Clariscan GE-Healthcare Pegylated starch 20 (USPIO) 36 n.a. VSOP-C184 Ferropharm Citrate 7 (VSPIO) 15���75 r1 =14 r2 =33.4 Gadoteridol (ProHance) Bracco Diagnostics, Inc ��� 1 (GBCA) 100 (umol (Gd) /kg) r1 =4 r2 =6 Currently available intravenous iron oxide nanoparticle contrast agents. Modified from Corot et al (2006). a Hydrodynamic diameter, laser light scattering. b Relaxometric properties (mM 1 sec 1) at 1.5 T, 37 1C, water or in plasma per mM Gd, or Fe. Superparamagnetic iron oxide nanoparticles in brain JS Weinstein et al 16 Journal of Cerebral Blood Flow & Metabolism (2010) 30, 15���35

USPIOs for imaging tumor neovasculature and assessing therapeutic response to antiangiogenic chemotherapeutic agents. Basic science review Ultrasmall Superparamagnetic Iron Oxide Particle Synthesis and Pharmacology The most common methods of USPIO synthesis for biomedical applications are coprecipitation of ferric and ferrous salts in an alkaline medium, with or without surface complexing agents such as dextran or polyethylene glycol, or microemulsion techniques with small amounts of iron ions trapped inside surfactant bubbles in an oil medium (Hartman et al, 2008 Laurent et al, 2008). The USPIOs consist of two components: an iron oxide core and a hydrophilic coating. It is the combination of these two compo- nents that determines their pharmacology. Passive targeting is perhaps most dependent on the hydro- dynamic radius and the surface charge (factors related to the coating material), as these character- istics determine circulation time, accessibility to tissues, opsonization, and rate of cell-type uptake (Thorek et al, 2006). Active targeting in the CNS takes advantage of nanoparticle surface modifica- tions (e.g., the addition of monoclonal antibodies or peptides) such as chlorotoxin, for glioma imaging. Current clinical trials primarily involve passive targeting. With respect to passive targeting, USPIOs behave differently than GBCAs for a number of reasons. First, because of their larger molecular size���up to 50 nm (compared with the 1nm gadolinium chelate), USPIOs extravasate much more slowly than standard GBCA, even in areas of severe BBB dysfunction (i.e., malignant glioma). Most GBCAs, in comparison, rapidly extravasate into the extravascular space in areas of BBB dysfunction and are rapidly cleared from the circulation via the kidneys. There are numerous formulations based on the gadolinium ion [Gd(III)], each with unique properties and safety profiles (Harpur et al, 1993). Ferumoxtran-10 is a first-generation USPIO that may have a slight advantage over ferumoxtyol for anatomic imaging of inflammatory lesions however, it is not safe for angiographic imaging procedures, which require a rapid infusion. Ferumoxytol was developed as an IV iron-replacement therapy, speci- fically for anemic patients with chronic kidney disease after bolus IV administration, it follows a useful distribution that is conceptionally based on the conservation of mass. Over time, a combination of events occur leading to contrast enhancement: USPIOs slowly leak across the BBB (mechanism incompletely understood) there may also be uptake of 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 oxide particles 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 enhancement intensity peaks B24 h after injection and can generally be visualized even 72 h after injection, although it is usually faint and more diffuse (Neuwelt et al, 2007). In contrast to GBCAs, there is no renal elimination of USPIO, which accounts for the enhanced safety profile in patients with renal dysfunction who appear to be at increased risk for contrast-induced nephropathy or nephrogenic sys- temic fibrosis (Neuwelt et al, 2008). Unlike USPIOs, the plasma half-life of SPIOs is on the order of minutes. This is due to their larger particle size ( 50 nm) and negative surface charge, which lead to rapid elimination. Iron-loaded mono- nuclear cells can actively cross a relatively intact BBB in cases of an active inflammatory process (Oude Engberink et al, 2007), but because of their large size, SPIOs do not leak across BBB defects like free USPIOs (as in malignant gliomas). In the clinical setting, FDA-approved doses of ferumoxides (SPIOs) were given to 20 patients with intrinsic or metastatic brain tumors. No enhancement was visualized in any patient at 30 mins or 4 h (Varallyay et al, 2002). The safety profile of clinically useful USPIOs varies based on their molecular structure. After cellular internalization, iron oxide nanoparticles accumulate in lysosomes in which the low pH breaks the iron oxide core down into iron ions. These ions are then incorporated back into the hemoglobin pool (Thorek et al, 2006). The type of coating used has a significant impact on the immunologic response to USPIOs to date, studies have shown that polymer- coated nanoparticles have minimal impact on cell viability and function (Bourrinet et al, 2006 Thorek et al, 2006). Trypan blue exclusion-based toxicity studies, using high concentrations of USPIOs, show good tolerance with minimal cell death. However, there have been reports of free radical generation, decreased cell proliferation, and even cell death with some 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 avoid mast cell degranulation, newer agents, such as SHU 555 C and ferumoxytol can be safely given as a rapid IV bolus. Ferumoxytol has been tested as an iron supple- ment therapy in patients with renal failure up to the dose of 510 mg (Landry et al, 2005 Spinowitz et al, 2005). For MRI contrast agent applications, the amount of iron administered (typically 4 mg/kg) is much lower than doses resulting in acute systemic toxicity (above 60 mg/kg) or chronic iron overload Superparamagnetic iron oxide nanoparticles in brain JS Weinstein et al 17 Journal of Cerebral Blood Flow & Metabolism (2010) 30, 15���35