Superparamagnetic Iron Oxide Nanoparticles in Biomedicine: Applications and Developments in Diagnostics and Therapy

Abstract

Superparamagnetic iron oxide nanoparticles (SPIO) can be used to image physiological processes and anatomical, cellular and molecular changes in diseases. The clinical applications range from the imaging of tumors and metastases in the liver, spleen and bone marrow, the imaging of lymph nodes and the CNS, MRA and perfusion imaging to atherosclerotic plaque and thrombosis imaging. New experimental approaches in molecular imaging describe undirected SPIO trapping (passive targeting) in inflammation, tumors and associated macrophages as well as the directed accumulation of SPIO ligands (active targeting) in tumor endothelia and tumor cells, areas of apoptosis, infarction, inflammation and degeneration in cardiovascular and neurological diseases, in atherosclerotic plaques or thrombi. The labeling of stem or immune cells allows the visualization of cell therapies or transplant rejections. The coupling of SPIO to ligands, radio- and/or chemotherapeutics, embedding in carrier systems or activatable smart sensor probes and their externally controlled focusing (physical targeting) enable molecular tumor therapies or the imaging of metabolic and enzymatic processes. Monodisperse SPIO with defined physicochemical and pharmacodynamic properties may improve SPIO-based MRI in the future and as targeted probes in diagnostic magnetic resonance (DMR) using chip-based µNMR may significantly expand the spectrum of in vitro analysis methods for biomarker, pathogens and tumor cells. Magnetic particle imaging (MPI) as a new imaging modality offers new applications for SPIO in cardiovascular, oncological, cellular and molecular diagnostics and therapy.

Key Points:

• SPIO can be used for diagnosis, MR imaging, and treatment.

• Monodisperse SPIO improve physicochemistry and pharmacodynamics.

• SPIO in targeted probes can be used in in-vitro diagnostic imaging (µNMR).

• The potential to use SPIO in magnetic particle imaging (MPI) must be evaluated.

Citation Format:

• Ittrich H, Peldschus K, Raabe N et al. Superparamagnetic Iron Oxide Nanoparticles in Biomedicine: Applications and Developments in Diagnostics and Therapy. Fortschr Röntgenstr 2013; 185: 1149 – 1166

Zusammenfassung

Mithilfe von Superparamagnetischen Eisenoxid-Nanopartikeln (SPIO) können physiologische Abläufe sowie anatomische, zelluläre und molekulare Veränderungen in Krankheitsprozessen abgebildet werden. Die klinischen Anwendungen reichen von der Tumor- und Metastasenbildgebung in Leber, Milz und Knochenmark, über die Lymphknoten- und ZNS-Bildgebung, die MR-Angiografie und Perfusionsdiagnostik bis hin zur atherosklerotischen Plaque- und Thrombosebildgebung. Die experimentellen Anwendungsfelder in der Molekularen Bildgebung umfassen neben ungerichteter SPIO-Anreicherung (Passive Targeting) in Entzündungsregionen und Tumoren sowie assoziierten Makrophagen die zielgerichtete Akkumulation von SPIO-Liganden (Active Targeting) an/in Tumorendothelien und Tumorzellen, Apoptose-, Infarkt-, Inflammations- und Degenerationsarealen in kardiovaskulären und neurologischen Erkrankungen, in atherosklerotischen Plaques oder Thromben. Die SPIO-Markierung von Stamm- oder Immunzellen erlaubt die Visualisierung von Zelltherapien oder Transplantat-Abstoßungen. Die Kopplung von SPIO an Liganden, Radio- und/oder Chemotherapeutika, die Einbettung in Trägersysteme oder aktivierbare Sonden sowie deren extern gesteuerter Fokussierung (Physical Targeting) ermöglicht molekulare Tumortherapien sowie die Abbildung metabolischer und enzymatischer Prozesse. Monodisperse SPIO mit definierten physikochemischen und pharmakodynamischen Eigenschaften könnten die SPIO-unterstützte MRT verbessern und zukünftig als zielgerichtete Sonden in der Diagnostischen Magnetresonanz (DMR) unter Nutzung von chipbasierten µNMR das Spektrum der In-vitro-Analysemethoden für Biomarker, Pathogene und Tumorzellen entscheidend erweitern. Magnetic Particle Imaging (MPI) als neues Abbildungsverfahren könnte SPIO sowohl in der kardiovaskulären, onkologischen, zellulären und molekularen Diagnostik und Therapie neue Anwendungsfelder eröffnen.

Key words

SPIO - molecular imaging - MRI - contrast media - iron oxide - nanoparticles

Introduction

The term superparamagnetic, first mentioned in the mid-1950 s [1], describes the ability of a ferrimagnetic or ferromagnetic material to be magnetized in the presence of an external magnetic field and to completely lose this magnetization once the magnetic field is deactivated (no remanent magnetization) [2]. This phenomenon is physically based on the Brown and Neel relaxation [3].

Nanoparticles typically refer to particles with a size of 1 – 100 nm, but the physical, chemical, and biological properties can differ greatly compared to macroscopic material with the same composition [4].

Superparamagnetic particles of iron oxide (SPIO) have an iron oxide core that is monomer- or polymer-coated/stabilized. The advantages of SPIO, such as good suspensibility, a highly reactive surface, uniform particle size distribution, and the possibility of additional coating modification by conjugating with specifically binding ligands (e. g. antibodies) has made it possible to use these particles in biomedicine since the mid-1970 s in immunomagnetic cell separation [5].

With the broad application of magnetic resonance imaging (MRI) in medical diagnostics starting in the mid-1980 s, another SPIO application field developed, namely use as a negative, i. e. signal-eliminating, contrast agent in MRI [6]. The nonspecific uptake of SPIO in the mononuclear phagocyte system (MPS) or reticuloendothelial system (RES) after intravenous application made it possible at the end of the 1980 s to use SPIO in preclinical and clinical diagnostic MRI of organs and organ systems, in particular the liver and spleen [7], and lymph nodes [8] and bone marrow [9]. As a result of systematic further development of SPIO, in particular size variation and coating modifications, numerous additional areas of application in preclinical and clinical MR diagnostics have been tested in the last two decades. Therefore, SPIO have been frequently tested in preclinical feasibility studies as a T1 contrast agent for MR angiography in vascular diagnostics [10], as a T2 or T2* contrast agent for the detection of atherosclerotic plaques [11] and vascular thrombi [12], in perfusion diagnosis of tumors [13], myocardium [14], renal parenchyma [15], vital brain [16], cerebral ischemia [17], and the placenta [18], and in the diagnosis of synovial structures, e. g. in the knee [19]. Newer applications in the primarily still preclinical experimental field of molecular imaging describe on a cellular level the in vitro labeling of cell types (e. g. macrophages, lymphocytes, progenitor/stem cells) with SPIO (cell labeling) and their diagnostic in vivo MR imaging and migration tracking (tracking/migration monitoring) [20] [21]. Additional studies on a molecular level describe the use of SPIO and specifically targeted SPIO conjugates (targeted probes) for labeling cell-surface molecules (e. g. cell receptors or antigens [22]) in combination with gene therapy, chemotherapeutic, or radiotherapeutic agents as a combined diagnostic-therapeutic agent (theranostics [23]) and tumor-thermoablative use in hyperthermia [24]. The embedding of SPIO in nanoencapsulations or microencapsulations with different controllable surface properties (e. g. in micelles or liposomes) recently made it possible to use SPIO in the imaging of metabolic processes (e. g. lipid metabolism [25] [26]). Moreover, the synthesis of novel, monodisperse SPIO with optimized physicochemical and pharmacodynamic properties allows more precise addressing of target structures, greater accumulation in the target area, improved target-environment contrast, and more exact parametric or quantitative SPIO-MRI.

In addition to the use of SPIO in diagnostic MRI and in the treatment of diverse diseases, use in a new tomographic imaging modality currently undergoing preclinical evaluation, namely magnetic particle imaging (MPI) [27], is being tested and optimized.

Based on the statements of Taupitz et al. [28], the present study is intended to provide a current overview of the morphological, physical-chemical, and biological characteristics of different SPIO, the current status in clinical application, and the current and future preclinical experimental application fields of SPIO in molecular imaging and treatment.

Particle types and particle properties

Definition and magnetic properties in MRI

Superparamagnetic particles of iron oxide (SPIO) are a separate class of MR contrast agents that have a size spectrum of only a few nanometers to several micrometers and influence the spin-spin (T2) and spin-lattice (T1) relaxation. After local accumulation, SPIO shorten the T2, T2* or T1 relaxation times of surrounding tissues. This causes a signal-reducing T2 and T2* effect (negative contrast) or a signal-increasing T1 effect (positive contrast). In general, the r₂ or r_2* relaxivity of SPIO as the reciprocal of the T2 or T2* relaxation (r₂ = 1/T2 or r_2* = 1/T2*) increases with an increasing core diameter, i. e., the signal-reducing effect in T2- and T2*-weighted sequences due to local dephasing in the tissue in the presence of SPIO (“susceptibility effect”) is greater at the same local concentration for SPIO with a high r₂ and r_2* than for SPIO with a low r₂ and r_2*. In contrast to this behavior, the T1-shortening effect, i. e., the signal-increasing effect in T1-weighted sequences, increases with a smaller SPIO core diameter. Consequently, SPIO with a large diameter are suitable for applications based on the signal reduction of SPIO in T2 and T2*-weighted sequences, while small SPIO are used as signal amplifiers in T1-weighted sequences. This relationship is described by the relaxivity ratio of a SPIO (r₁/r₂), i. e., SPIO with a low relaxivity ratio are to be used as a T2 / T2*/negative contrast agent and SPIO with a high relaxivity ratio as a T1/positive contrast agent. Measured on the basis of the absolute concentration of particles in tissue, SPIO in T2 and T2*-weighted images result in a greater negative contrast (detectability mmol-µmol) [29] than comparable concentrations of paramagnetic gadolinium (Gd)-containing contrast agent (detectability to cmol-mmol), making SPIO particularly preferred for use in molecular imaging.

With respect to the selection of MR sequences and sequence parameters, it must be stated that gradient echo sequences (GRE, FFE) are more sensitive with respect to magnetic susceptibilities than spin echo sequences (SE) and the sensitivity of the sequence increases with the decrease of the flip angle (FA< 20°), extension of the echo time (TE> 10 – 20 ms) and repetition time (TR> 100 ms), and the increase of the spatial resolution (reduction of the partial volume effect) [30]. In addition to these negative-contrast techniques, newer developments in susceptibility-weighted imaging (SWI) use magnitude information as well as phase information from the complex data of spatially highly resolved 3 D gradient echo sequences to generate tissue imaging with improved contrast without using a contrast agent [31] [32] [33] or a positive-contrast image after the administration of a contrast agent (e. g. SPIO) [34] [35]. Using suitable post-processing algorithms, susceptibility/phase gradient maps (SGM/PGM) can be created by using voxel-based phase information and creating mask images [35] [36]. As a result, the contrasts in heterogeneous tissues (e. g., between gray and white brain matter) for the detection of tissue iron deposits or venous blood vessels, as well as the sensitivity to paramagnetic and superparamagnetic substances or contrast agents can be improved. It was able to be shown that these techniques in addition to increasing intrinsic tissue contrasts are also suitable for the detection of the smallest quantities of SPIO (e. g. SPIO-labeled cells), in particular in inhomogeneous tissues or structures with a low signal intensity (e. g. connective tissue) [34].

Particle structure, particle synthesis, and quality control

As a rule, SPIO are comprised of a crystalline core and a surrounding core-stabilizing and optionally aggregation-preventing coating whose physical-chemical and associated biological properties can be varied in virtually any way desired ([Fig. 1]). The crystalline core of SPIO is comprised of ferri(Fe^3 +)magnetic and ferro(Fe^2 +)magnetic material in the form of maghemite (γFe₂O₃) and magnetite (Fe₃O₄) and is synthesized in usual protocols with controlled precipitation of iron oxides in aqueous solution (coprecipitation of iron salts by adding a base, e. g. sodium hydroxide, ammonium hydroxide, < 100 °C) [37] or in organic solution (high temperature decomposition of iron acetylacetonate in phenyl ether, alcohol, oleic acid or oleylamine [38], 250 – 300 °C). Since it is difficult to control particle size (in particular < 20 nm) and to achieve ideal crystallinity and monodispersity (standard deviation of the particle diameter 5 – 10 %) in an aqueous solution and the biological properties of monodisperse SPIO and SPIO conjugates with respect to biodistribution, bioelimination, and contrast behavior can be better controlled, which is necessary for targeted use in molecular imaging, high-temperature decomposition synthesis has been increasingly used in recent years [39]. The thus-created cores have a size of a few (2 – 3) nanometers to multiple tens (20 – 30) of nanometers ([Fig. 2]). The synthesizability and stability of SPIO cores decreases with an increasing core diameter so that these can only be reliably created up to a size of approximately 30 – 40 nm. The coating is then applied by adding a stabilizing base coating material (e. g., citrate, dextran, carboxydextran, chitosan, pullulan, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyethylenimine (PEI), polyethylene oxide (PEO), polysaccharide, albumin, lipids, etc.) to monocrystalline or polycrystalline SPIO. For in vivo use, organically synthesized SPIO must be transferred to aqueous media by depositing hydrophilic outer coatings. In further steps the particle coating can be secondarily modified, e. g. by the covalent binding of ligands (e. g. proteins, antibodies, etc.). With respect to particle size distribution (dispersity), monodisperse particles, i. e., uniform SPIO with close particle size distribution, can be differentiated from polydisperse, polymorphous SPIO with significant size variance depending on the synthesis.

Fig. 1 Schematic structure of a SPIO. In arrangement with randomly aligned (without external magnetic field) additional magnetic domains (middle), the magnetic domain of an iron oxide crystal (left) forms a SPIO crystal/core, which can then be coated with a stabilizing coating material to form a monocrystalline SPIO (upper right) or polycrystalline SPIO (bottom right) (modified according to Wang et al. [34]).

Fig. 2 Superparamagnetic iron oxide particles compared with respect to size and dispersity. Polydisperse SPIO such as a Resovist^® (Bayer Healthcare Pharmaceuticals, 50 – 100 nm, upper left, scale 100 nm) and b Sinerem^® (Guerbet) upper right, 20 – 40 nm, scale 200 nm) manufactured by coprecipitation synthesis in aqueous solution (TEM: R. Reimer/H. Hohenberg (HPI Hamburg)), c–f monodisperse USPIO from high temperature decomposition in organic solution, c SPIO cores (10 ± 1 nm) before and d after PMAcOD coating (~20 nm, scale 50 nm, TEM: R. Reimer/H. Hohenberg (HPI Hamburg)), e, f commercially available monodisperse polymers SPIO from Ocean NanoTech (www.oceannanotech.com), e SHP-15 (15 ± 1.5 nm, scale 50 nm) and f SHP-40 (40 ± 4 nm).

Size isolation or filtration of the preferred SPIO can be achieved by column chromatography, centrifugation and/or dialysis. Quality control with respect to morphology, core/coating size, dispersity, and crystallinity can be accomplished using transmission electron microscopy (TEM), X-ray diffraction (XRD), and dynamic light scattering (DLS). The magnetic properties can be determined via nuclear magnetic resonance spectroscopy (NMR) and the iron content via atomic absorption spectroscopy (AAS).

Particle size and coatings

SPIO are divided into different subgroups according to size [40]. With respect to size, a differentiation must be made between the size of the iron oxide core, the total size of the particle with coating, and the total particle size in aqueous solution after the deposition of water molecules (hydrodynamic diameter). The resulting data regarding particle size in vitro can change when used in vivo due to the additional deposition of salts, opsonization with plasma proteins (e. g. albumin), lipids or carbohydrates (particle corona) depending on the surface charges of the coating molecules, or due to the clustering of SPIO to form SPIO conglomerates so that the total size in vitro can differ greatly in some cases.

The first iron oxide particles “originally” referred to as SPIO have a size of 40 – 150 nm and are referred to as standard SPIO (SSPIO) (e. g. ferumoxide (AMI-25 (Endorem^®/Ferridex^®)), ferucarbotran (SHU 555A (Resovist^®)). Comparatively, SPIO of approximately 20 – 40 nm are referred to as ultrasmall SPIO (ultrasmall superparamagnetic iron oxide – USPIO) (e. g. ferumoxtran-10 (AMI-227 (Sinerem^®/Combidex^®)), ferucarbotran (SHU 555C (Supravist^®)), (Code 7228 (Rienso^®)), PEG-feron (NC100 150 (Clariscan^®)) or FeO-BPA. Even smaller SPIO of a size of approximately 15 – 20 nm with a polymer coating are classified under consideration of the monocrystalline structure as MION (monocrystalline iron oxide nanoparticles) [41]. The smallest currently synthesizable SPIO have a core of 3 – 4 nm and a monomer coating (e. g. citrate) so that total particle sizes of only a few nanometers (approximately 5 – 7 nm) can be realized and are referred to as very small iron oxide particles (VSOP) [42] [43] [44].

By cross-linking the coating materials, larger SPIO complexes that can reach a size of several micrometers and be classified as CLIO (cross-linked iron oxide) can also be created [45]. Moreover, the formation of larger SPIO particle constructs by embedding SPIO in different coating systems (e. g. micelles or liposomes) is currently being tested [25]. As a result, on the one hand clustering of the SPIO in a small space with an increase in the R2 / R2* relaxation rate is achieved and on the other hand the surface properties of natural biological transport systems are used or imitated in order to image their distribution processes noninvasively. This includes the embedding of SPIO in lipoprotein-lipid coatings (nanosomes [25] [26]) or the embedding of oleic acid-stabilized SPIO and optionally additional therapeutic agents in cross-linked phospholipid nanoemulsions referred to as colloidal iron oxide nanoparticles (CION). With the latter construct, a platform for simultaneous imaging and treatment (theranostics) is proposed [46]. An overview of the SPIO currently used in clinical application and testing is provided in [Table 1]. SPIO of different size, shape, dispersity, coating, and functionalization can currently be commercially procured from different manufacturers for experimental, preclinical testing. [Table 2] provides an overview of several manufacturers.

Table 1 Superparamagnetic iron oxide particles in clinical application and clinical and preclinical evaluation (modified according to [43] and [28]). Deviations in the information regarding r₁ and r₂ are possible depending on the source

name/reference	generic name	registered name	core	contrast	type	core size/ hydrodyn. diameter [nm]	coating	r1 [mMol-1 ls-1]*	r2 [mMol-1 ls-1]*	r1 r2	blood half-life(T1/2)	concentration	indication	application/development phase	manufacturer/distributor
AMI-25 [34] [195] [196] [197] [198] [199] [200]	Ferumoxide	Feridex®, Endorem®	Fe3 +/Fe2 +	T2	SPIO	5 – 6 80 – 150	Dextran	30[1]+9.9[2]	1001 1582	0.31 0.072	8 min.	11.2 mg Fe/ml	liver, spleen, bone marrow	approved[3]	AMAG PharmaceuticalsGuerbet
SHU 555A[196] [198] [199] [201] [202] [203]	Ferucarbotran	Resovist®, Cliavist®	Fe3 +/Fe2 +	T2	SPIO	4.2 50 – 100	Carboxy-dextran	25.41 9.72	1511 1892	0.1710.052	10 min.	0.5 mmol/l	Liver, spleen, bone marrow	approved[4] EU, Japan, Australia, (ØUSA)	Bayer Healthcare Pharmaceuticals
AMI-227 [34] [95] [196] [197] [202] [204]	Ferumoxtran-10	Sinerem®, Combidex®	Fe3 +/Fe2 +	T2,(T1)	USPIO	4 – 6 20 – 40	Dextran	311 19.52	781 87.62	0.391 0.222	24 – 30 h	20 mg Fe/ml	MRA, liver, spleen, bone marrow	phase III[5]	GuerbetAMAG Pharmaceuticals
P904 [74] [168]	–	–	Fe3 +/Fe2 +	T1, T2	USPIO	4 – 6 21	Amino-alcohol- glucose-derivative	142	872	0.162	3.5 h		MRA, atherosclerosis, adiposity	preclinical	Guerbet
SHU 555C[81] [203]	Ferucarbotran	Supravist®	Fe3 +/Fe2 +	T2,(T1)	USPIO	3 – 4 20 – 30	Carboxy-dextran	181 112	411 382	0.441 0.292	6 – 8 h	0.5 mmol/l	MRA, lymph nodes	phase II[6]	Bayer Healthcare Pharmaceuticals
Code 7228 [159] [197] [202]	Ferumoxytol	Feraheme® Rienso®	Fe3 +/Fe2 +	T1	USPIO	6 – 7 28 – 32	Carboxymethyl-dextran	382	832	0.462	10 – 14 h	30 mg Fe/mL	MRA	approved for iron deficiency anemia	AMAG Pharmaceuticals, Takeda Pharmaceutical Company Limited
NC100150 [34] [196]	FeruglosePEG-feron	Clariscan®	Fe3 +/Fe2 +	T2, T1	USPIO	4 – 7 11 – 20	Starch/PEG	201	351	0.571	6 h	29.8 mg Fe/mL	MRA, lymph nodes, liver, spleen	phase III	GE Healthcare
FeO-BPA [91]	–	–	Fe3 +/Fe2 +	T1	USPIO	5 – 7 20	Oxidized starch	202	352	0.572	45 – 100 min.	–	MRA	preclinical
MION-46 L[205] [206] [207] [208]	–	–	Fe3 +/Fe2 +	T2,(T1)	MION	4 – 6 8 – 20	Dextran	16.51	34.81	0.471	> 10 h	–	MRA, lymph nodes, tumor, infarction	preclinical
VSOP-C184 [36] [37] [196] [207] [208] [209] [210]	–	–	Fe	T1	VSOP	4 – 5 7 – 9	Citrate	191 142	291 33.42	0.661 0.422	30 – 60 min.	0,5 mmol/l	MRA	phase I	Ferropharm GmbH
AMI-121 [211]	Ferumoxil	GastroMARK®, Lumirem®	Fe3 +/Fe2 +	T2	SPIO	10> 300	Silicon	3.4[7]	3.87	0.897	< 5 min.	52.5 mg Fe/300 mL	gastrointestinal	approved	AMAG Pharmaceuticals Guerbet
OMP [34]	Ferristene	Abdoscan®	Fe2 +	T2	SPIO	50 3 000 – 3 500	Styrol/divinylbenzene	no data	no data	no data		23.4 Fe/200 ml	gastrointestinal	approved in EU to 2002, (ØFDA)	GE Healthcare

¹ Measured at 0.47 T, 37 °C.

² Measured at 1.5 T, 37 °C.

³ No longer produced and distributed since 2011.

⁴ No longer distributed in Europe since 2009, only available in Japan (international pharmacy).

⁵ Application for clinical approval from EMEA withdrawn in 2007.

⁶ No longer being developed.

⁷ Measured at 0.97 T, 37 °C

Table 2 Selection of some of the manufacturers of experimental SPIO

manufacturer	location/head office	website
Bangs Laboratories, Inc.	9025 Technology Drive Fishers, IN 46 038 – 2886 USA	www.bangslabs.com
Centrum für Angewandte Nanotechnologie (CAN) GmbH	Grindelallee 117 20 146 Hamburg Germany	www.can-hamburg.de
Chemicell GmbH	Eresburgstraße 22 – 23 12 103 Berlin Germany	www.chemicell.com
Ferropharm GmbH	Potsdamer Str. 18a 14 513 Teltow Germany	www.ferropharm.de
Kisker Biotech GmbH & Co. KG	Postbox 1329 48 543 Steinfurt Germany	www.kisker-biotech.com
MagForce AG	Max-Dohrn-Str. 8, Haus B 5.2 10 589 Berlin Germany	www.magforce.de
Micromod Partikeltechnologie GmbH	Friedrich-Barnewitz-Str.4 18 119 Rostock Germany	www.micromod.de
Miltenyi Biotec GmbH	Friedrich-Ebert-Straße 68 51 429 Bergisch Gladbach Germany	www.miltenyibiotec.com
NanoPET Pharma	Robert-Koch-Platz 4 10 115 Berlin Germany	www.nanopet-pharma.com
Ocean Nanotech	2143 Worth Lane Springdale, Arkansas 72 764 USA	www.oceannanotech.com
Phosphorex, Inc.	94 South Street Hopkinton, Ma 01 748 USA	www.phosphorex.com
Spherotech, Inc.	27 845 Irma Lee Circle, Unit 101 Lake Forest, IL 60 045 USA	www.spherotech.com

Distribution, degradation and toxicity

The size and surface properties (in particular charge) have a decisive influence on the elimination, cell response, and toxicity of SPIO [47]. Thus, SPIO with a small hydrodynamic diameter and neutral and hydrophilic surface are opsonized and phagocytized more slowly than large SPIO with an ionic and hydrophobic coating. SSPIO are phagocytized or endocytized by monocytes, macrophages, or oligodendroglial cells of the RES and are thus removed from the blood stream so that they have a comparably short blood half-life of a few minutes [48] (AMI-25: 8 min., SHU-555A: 10 min.) [49]. SSPIO accumulate primarily in the liver (80 – 90 %), spleen (5 – 8 %), and bone marrow (1 – 2 %) [49]. In comparison, USPIO and VSOP have a longer blood half-life which makes it possible to use these as blood pool contrast agents (e. g. blood half-life AMI-227: 200 min., SHU 555C: 6 – 8 h, VSOP-C184: 30 – 60 min.) [49]. Small particles of less than 5 nm can pass through the glomerulus and be eliminated renally [50].

After uptake of the SPIO in autochthonous, specialized macrophages of the liver (Kupffer cells), these are degraded lysosomally [51], and the core material is supplied to the iron storage pool of the body (total body iron approximately 4 – 5 g) and deposited in the liver in the form of ferritin and/or hemosiderin [52]. The natural decomposition of these iron reserves is only possible via hematopoiesis (Fe^2 + as central atom in the hemoglobin) and the exfoliation of epithelial cells of the skin and bowel. The coating material is eliminated via other decomposition and elimination paths, e. g. the coating of ferumoxtran-10 (Sinerem^®) is degraded via intracellular dextranases and is eliminated primarily renally (89 % in 8 weeks) [53].

It has been able to be shown experimentally in vitro that polymer-coated SPIO (PEG/dextran-SPIO) only have a minimal effect on cell function and vitality [54]. However, newer studies show that internalized SPIO can influence the cell expression pattern (e. g. by oxidative stress), cell proliferation, and cell differentiation [55]. Methods for core labeling (e. g. ⁵⁹Fe [56]) and coating labeling with radioisotopes [57] (e. g. ¹¹¹In [58], ⁶⁴Cu [59], ^99 mTc [60] or ¹⁴C) and quantitative evaluation methods have been proposed to clarify the SPIO metabolic processes, i. e., in vivo biodistribution and biodegradation of the core and coating material, prior to a potential clinical application, particularly to clarify any possible nanoparticle toxicity.

In diagnostic concentrations (20 – 50 mg Fe), no toxic side effects after i. v. injection of SPIO have been observed to date [61]. Headache or back pain, vasodilation, hypotension, or allergic reaction in the form of urticaria can occur on rare occasions [62]. No systemic toxic effects have been observed in animal experiments at concentrations up to 100 mg Fe/kg [63]. The amount of a chronic iron overload (> 20 g) is not reached even when used multiple times.

SPIO in clinical application

The SPIO currently in clinical application are used in MR imaging to take advantage of their T2/T2*/T1-shortening effect. Blood elimination occurs nonspecifically via natural phagocytic elimination mechanisms, so-called “passive targeting”. The accumulation of SPIO in tissue is used for T2/T2* imaging, the T1 effect during blood circulation. The blood half-life and the phagocytic elimination by the RES are largely influenced by the particle size (hydrodynamic diameter) and the surface properties (chemical composition/charge) of the SPIO.

T2/T2* applications

Liver imaging

SPIO with a hydrodynamic diameter of 100 – 150 nm (SSPIO) are nonspecifically absorbed by Kupffer cells (autochthonous macrophages) of the normal liver parenchyma resulting in a loss of signal of the liver tissue in T2w and T2*s MRI. Primary liver malignancies (hepatocellular carcinoma (HCC), cholangiocellular carcinoma (CCC)) or secondary liver malignancies (metastases) do not have any Kupffer cells so that there is no SSPIO accumulation in these lesions and no T2w/T2*w signal loss [64] ([Fig. 3]). Consequently, the lesion-to-liver contrast increases. This increases the sensitivity for detecting tumorous lesions [65] and can also improve the classification of liver tumors and the grading [66] and delimitation of an HCC with respect to benign liver tumors or hyperplastic nodes [67]. The two SSPIO available in clinical use were ferumoxide (Endorem^®, Guerbet) [68] and ferucarbotran (Resovist^®, Bayer Healthcare Pharmaceuticals) [69]. However, the production and sale thereof have been discontinued since 2011 for Endorem^® and 2009 for Resovist^® in Europe due to the small sales market and the introduction of the hepatobiliary Gd-based contrast agent, Gd-EOB (Primovist^®, Bayer Healthcare Pharmaceuticals), with an improved ability to detect liver lesions [70]. Resovist^® is only still sold on the Japanese market and can be procured there (international pharmacy).

Fig. 3 Liver with multifocal HCC a T1w MRI 5 min. after i. v. application of Gd-DTPA and b T2*w MRI before and c after i. v. administration of SPIO (ferucarbotran (Resovist^®)). Accumulation of SPIO in Kupffer cells of the liver results in a significant signal loss in the normal, healthy liver parenchyma with a lack of signal reduction in the HCC lesions (white arrows).

To avoid nephropathy or the risk of nephrogenic systemic fibrosis (NSF) when using a Gd-containing contrast agent in patients with limited kidney function (glomerular filtration rate (GFR) < 30 ml/min. per 1.73 m²), SPIO can be a safe alternative for detecting and differentiating liver lesions [71].

Spleen imaging

Analogously to the liver, the accumulation of SPIO in spleen macrophages improves the detection of metastases or rare primary spleen tumors by increasing the lesion-to-spleen contrast [65]. Another application field is the diagnosis of heterotopic splenic tissues, such as an accessory spleen or splenosis. In this case, SPIO can aid differentiation with respect to pancreatic tumors (intrapancreatic accessory spleen) or peritoneal metastases (splenosis after splenectomy or abdominal trauma) due to the accumulation in these lesions [72].

Lymph node imaging

Smaller SPIO (USPIO, hydrodynamic diameter ~30 nm) can be used for MR lymphography and for lymph node staging following intravenous or local subcutaneous injection. Due to the longer circulation time of dextran-coated USPIO as a result of the lower accumulation in the RES of the liver and spleen and the small size of USPIO compared to SSPIO, these can exit the vascular system after intravenous application (extravasation), are transported lymphogenically, and are absorbed physiologically in the lymph nodes by macrophages, resulting in a signal loss of the lymph nodes in T2w and in particular T2*w gradient echo sequences. Depending on the extent of the lymph node invasion, metastatic lymph nodes exhibit this signal loss only partially or not at all as could be shown in clinical studies in carcinomas of the head-neck [73], breast [74], bronchus [75], esophagus [76], stomach [77], rectum [78], and in pelvic/urological carcinomas [79], and can therefore result in an improvement of the lymph node staging. However, USPIO are currently not approved for clinical application. Due to the not definitively proven improvement of the specificity of MR for the detection of metastatic lymph nodes using Sinerem^® in a main study in phase III (MR lymph node analysis in pelvic carcinoma patients), the application for clinical approval of Sinerem^® from the EMEA was retracted by Guerbet in 2007 (doc. ref. EMEA/595 122/2007). A further USPIO development with a similar application spectrum called P904 is currently undergoing preclinical evaluation [80].

Tumor imaging

SPIO can cause signal reductions in T2w and T2*W pulse sequences in tumor regions due to the direct uptake in tumor cells and tumor-associated macrophages or via the increased extravasation in tumor-associated vessels with increased capillary leakiness. This was able to be observed clinically in liver metastases [81] and was shown experimentally in gliosarcomas and gliomas [82] [83].

Bone marrow imaging

As in the macrophages in the liver and spleen, the increased uptake in phagocytic cells of bone marrow results in a signal loss in T2w or T2*w pulse sequences [9]. The lack of focal or diffuse signal loss signalizes an infiltration by malignant cells also in this case [84]. In addition, differences in the signal behavior of inflammatory medullary cavity lesions (osteomyelitis) and irradiated, hematopoietic, or reconverted bone marrow compared to normal or metastatic bone marrow after SPIO application are described [85].

Imaging of the gastrointestinal tract

Non-degradable and non-absorbable ferrofluids with large diameters (0.3 – 3.5 µm) can be applied after oral administration for suppression of the water signal of the bowel (Lumirem^®/GastroMARK^® (Guerbet/AMAG Pharmaceuticals), Abdoscan^® (GE Healthcare). This can be used to prevent overlapping in magnetic resonance cholangiopancreatography (MRCP), for better delimitation with respect to structures adjacent to the bowel (e. g. lymph nodes or uterus) and the evaluation (postoperative) of anatomical changes to the bowel [86], as well as for the detection of inflammatory bowel lesions after i. v. administration [87].

CNS imaging

The suitability of SPIO for MRI of the CNS was able to be shown in neurovascular (infarction [88]), neuro-oncological (tumors [89]) or neuroinflammatory processes (multiple sclerosis (MS) [90]) as well as for angiography and perfusion diagnosis [91]. Increased accumulation of SPIO in the microglia of the CNS in the case of disruption of the blood brain barrier (BBB) in tumorous CNS lesions [89] or inflammatory CNS lesions [92] which can be imaged with the help of T2w/T2*w MR sequences is observed in the case of the former applications.

Characterization of atherosclerotic plaques

Soft plaques, which are currently known to have a predisposition for acute atherothrombotic vascular occlusion and are vulnerable due to the high lipid content and the thin fibrous cap, have an increased macrophage content and efficient SPIO labeling is the goal of T2w and T2*w MR imaging of atherosclerotic lesions ([Fig. 4]). As a result, the increased accumulation of USPIO in atherosclerotic plaques could be proven both preclinically [11] [80] [93] and clinically [94], in particular in carotid plaques [95], in numerous studies but has not yet been widely used clinically.

Fig. 4 Atherosclerotic plaques a before and b after SPIO. After i. v. application of SPIO, there is signal loss of inflammatory atherosclerotic plaques (arrows) due to the accumulation of SPIO in plaque-associated macrophages (transverse slice orientation through the aortic arch, experimental in hereditary hyperlipidemic Watanabe (WHHL) rabbits, T-trachea). Osseous structures such as vertebral bodies/arches and ribs also show an increased signal loss (arrow tips) due to accumulation of SPIO in macrophages of the bone marrow.

T1 applications

Magnetic resonance angiography (MRA)

The relaxivity ratio (r₁/r₂), which increases as the particle diameter decreases, the sufficiently high r₁ relaxivity, and the comparatively long intravasal circulation times of USPIO and VSOP (blood pool contrast agent) allow application as a T1 contrast agent in luminographic MR vascular diagnostics (magnetic resonance angiography (MRA)). Numerous preclinical and several clinical studies describe their use both in MRA of individual vascular territories, such as pulmonary arteries [96], the aorta [80] [97], coronary arteries [10] [98] [99], renal arteries [10], mesenterial and portal veins [100], hepatic veins [101], and the inferior vena cava [102], as well as in whole-body angiography of arteries and veins for assessing stenoses and thromboses [103]. Potential USPIO for clinical application are Supravist^® (SHU 555C, phase I, Bayer Healthcare Pharmaceuticals), AMI-227 (Sinerem^®, phase III), and VSOP-184 (phase I). However, the dominance of the Gd-based contrast agent, which is quickly eliminated renally and has a safe application profile, has prevented the broad use of USPIO in MRA. In addition to contrast-free angiography techniques [104], contrast-enhanced MRA using USPIO and VSOP could be used as a safe alternative to Gd-containing contrast agents in patients with limited renal function (low GFR) to prevent contrast-induced nephropathy or the risk of NSF [71].

Thrombosis diagnosis

Luminographic T1w MR techniques using long-circulating blood pool SPIO (USPIO and VSOP) make it possible to diagnose thromboses in venous vessels that can be visualized indirectly as contrast-free areas in venous MRA [103] or experimentally with direct thrombus visualization [12].

Perfusion diagnosis

The T1 and T2 / T2* effect of USPIO and VSOP can be used in dynamic contrast-enhanced MRI (DCE-MRI) and dynamic susceptibility-weighted MRI (DSC-MRI) and the T1 and T2 / T2* effect of long-circulating USPIO can also be used in steady state free precision MRI (SSFP-MRI) for perfusion diagnosis of organs or organ lesions. The microvascular permeability [105] can be determined via T1w DCE-MRI during the wash-in phase, and the fractional plasma volume in tissues/tumors, which is closely correlated with the vessel density and the vessel size index, can be determined via SSFP-MRI via the change in the R2 / R2* relaxation rates before and after USPIO application (ΔR2 or ΔR2*) [106]. These noninvasively determined parameters can show the effectiveness of antiangiogenic or antitumoral treatments prior to a change in tumor size [107]. Organ and lesion perfusion diagnosis via USPIO and VSOP could be shown using the example of the heart [99], liver [108], spleen [65], and kidneys [109].

SPIO in preclinical and experimental application

Molecular imaging

Due to their variable size, the excellent signal properties in T2w and T2w* MRI, the possible coating variations and the consequently diverse conjugation options or functionalizations (targeting molecules), the simple biodegradation (iron metabolism), the ability to label with fluorescent dyes, radioisotopes, and therapeutic agents, and the simple traceability in correlative analysis procedures (light/electron microscopy), small SPIO (USPIO and MION) are suitable for use in molecular imaging for synthesizing targeted molecular probes (active targeting). Therefore, the spectrum of applications for SPIO and their derivatives has expanded continuously in recent years. Initial proof-of-concept experiments have helped different application areas for SPIO to become established in the last decade and some have found their way into clinical studies (e. g. SPIO cell labeling).

SPIO conjugation with antibodies and antibody fragments

The conjugation of SPIO with specific ligands, such as antibodies, peptides, polysaccharides, nucleotides, aptamers, and other synthetic mimetics, for generating targeted vectorized contrast agents (targeted SPIO) theoretically offers virtually unlimited applications in MR imaging of tumors, inflammation, cardiovascular, neurovascular, or degenerative diseases with close parallels to the developments in nuclear medicine (radioactively labeled ligands) [110].

Particularly in regard to antibodies (AB), SPIO can be conjugated with complete antibodies (SPIO-AB), monovalent antibody fragments, diabodies, triabodies, tetrabodies, single-chain antibodies, or minibodies [111]. If most successful studies were performed at high field strengths of up to 7 T, their translation to 1.5 T is also described [112]. Limitations on the use of SPIO-AB are currently the limited sensitivity of MRI, in particular in the case of low-expression target structures (low antigen density/receptor density, usually 1 – 100 nmol) and the small SPIO size (usually USPIO) needed for a long circulation time, extravasation capability, and tissue penetration. Currently successful concepts for increasing the specific accumulation of SPIO-AB are based on the receptor-mediated uptake of SPIO-AB conjugates in the target cells (intracellular trapping) [113] or on a 2-step strategy in which a target-recognizing biotinylated antibody and then a streptavidin-conjugated SPIO are injected [114]. The extremely high biotin-(strept)avidin binding affinity ensures accumulation of the conjugated SPIO in the target tissue.

Tumor imaging

In tumor imaging, antibody-SPIO conjugates were able to be used to successfully image numerous surface markers overexpressed in tumor vessels and cells, such transferrin receptor [115], folic acid receptor [116], VEGF [117], RGD [118], α_νβ₃-integrin as angiogenesis marker [119], Her2/neu (c-erb B-2) tyrosinase receptor [114], uMUC-1 [120], LHRH-R [121], EGFR [122], CEA [123], CEACAM5 ([Fig. 5]) [124], CXCR4 [125], CD20 [126] or B220 + (B-cell lymphomas) [127] with the help of T2w or T2*w MRI. An alternative, older approach is SPIO labeling of non-tumorous target structures for “inverse” imaging of tumors as was successfully shown in liver cells in an animal model (asialoglycoprotein(AGP) receptor) for the detection of liver neoplasias [128] or in pancreas cells (cholecystokinin A(CCKA) receptor) for the identification of pancreas tumors [129].

Fig. 5 Molecular tumor imaging with SPIO-conjugated antibodies. T2*w MRI of C57BI/6-SCID mice with FEMX-I tumors (CEACAM expression) of the left flank before (left) and after (right) i. v. administration of SPIO (upper row) shows minimal signal loss in the tumor periphery 12 h after application (arrows), caused by non-specific SPIO accumulation in tumor-associated macrophages. After the application of targeted T84.1-SPIO conjugates (bottom row), there is significant signal loss primarily in the highly vascularized tumor periphery due to specific AB-SPIO tumor binding (arrow tips), from Pöselt et al. [117].

Apoptosis imaging

Cell apoptosis plays an important role in numerous neoplastic, neurodegenerative, inflammatory, and cardiovascular-ischemic diseases. Successful MR imaging of apoptotic cells in vivo was able to be performed using targeted SPIO against phosphatidylserine after the conjugation of SPIO with the C2 domain of synaptotagmin I [130], annexin V [131], or annexin A5 [132].

Cardiovascular imaging

Expanding the spectrum of MR imaging using non-targeted USPIO already performed in clinical studies, specific, targeted USPIO were used in an attempt to address target structures in cardiovascular diseases, such as atherosclerosis, thrombosis, and myocardial infarction, to increase the T2w/T2w* signal-reducing effects via specific accumulation of the targeted SPIO and to thus detect earlier stages of the diseases.

As a result, apoptotic foam cells [133], oxidation-specific epitopes (malondialdehyde-lysine-epitopes of MDA-LDL [134]), oxidized phospholipid epitopes [134], scavenger receptors type A (with sulfated, dextran-coated SPIO) [135], VCAM-1 [136], E- and P-selectin [136], or oxidated LDL [137] in vulnerable and inflammatory plaques were able to be successfully labeled.

In molecular myocardial infarction imaging, the use of MION-conjugated antimyosin-F_ab (MION-R11D10) against myosin for labeling an infarcted myocardium is described [138].

In thrombus diagnosis, target structures in thrombuses, such as α₂β₃-intergrin [139], of activated thrombocytes [140] or activated coagulation factor XIII (FXIIIa) [141] were able to be successfully addressed.

Cell labeling and cell imaging in MRI

The ex vivo loading or labeling of cells with SPIO (cell labeling) allows the in vivo imaging and tracking (migration monitoring) of SPIO-labeled cells in T2w and T2w* MRI after application ([Fig. 6]).

Fig. 6 Cellular imaging of SPIO-labeled mesenchymal stem cells (MSC). a Fluorescence microscopy and b native light microscopy of a singular MSC after labeling with coating-fluorescence-labeled SPIO (cell core after DAPI staining, blue). c In vitro T2*w MRI of different quantities of SPIO-labeled MSC embedded in gelatin. d In vivo T2*w MRI and corresponding relaxometric T2* map (in ms) of SPIO-labeled MSC in the renal cortex of the left kidney (glomeruli) after selective application in the left renal artery.

Efficient ex vivo labeling of cells can be achieved by coating modifications, e. g. by coating with liposomes [142], by using lectins [143], by conjugating antigen-specific monoclonal antibodies (MION-46 L [144]), or by binding specific peptide chains (HIV-1 Tat peptide) to the dextran coating of SPIO (MION-Tat, CLIO-Tat [145]). Moreover, the use of transfection media [146], polyamines, lipids, or dendrimers [147] can result in enhanced cellular SPIO incorporation. Due to the partially complicated labeling protocols, simplified strategies using clinically approved materials (e. g. Endorem^®/Feridex^®, Superfect^®, Lipofectamine Plus^®, poly-L-lysine (PLL), protamine) and low cell toxicity have been described in recent years [148] [149]. While most studies do not describe any significant changes in cell behavior (e. g. apoptosis rate, proliferation index, differentiation behavior) [150] [151], individual studies show changes in migration behavior and the ability to form colonies [152], in cell vitality [153], in differentiation behavior [154], or in the expression pattern [152]. By the same token, the influence of cell labeling on the long-term behavior of cells and the course of disease is currently unclear [155] and must be studied more closely in the future [156].

Successful in vivo detection and migration monitoring of SPIO-labeled cells was able to be demonstrated in numerous studies, e. g. in implanted hematopoietic, mesenchymal, or neuronal stem cells in the CNS [157], heart [158], liver [159], spleen [160], bone marrow [160], kidneys [161], joints [162], and muscles [163], endothelial progenitor cells [164], transplanted islet cells [165], and lymphocytic and moncytic cells (natural killer cells in oncological cell therapies [166]). Moreover, the migration of macrophages in apoptotic tissue replacement [167], tumors [168], or vascular aneurysms [169] was able to be visualized in vivo. With respect to in vivo sensitivity, it is possible under ideal conditions to detect single SPIO-labeled cells [170].

Transplant diagnostics, imaging of rejection reactions (graft rejection)

The increased local presence of macrophages and the associated increased accumulation of SPIO during a rejection and changes in the permeability of transplant vessels were able to be imaged in MRI in an animal model during the rejection of heart [171] and kidney transplants [172].

Inflammation, infection, and adiposity imaging

As in the imaging of rejection reactions, SPIO also accumulate in the macrophages of inflammatory lesions. Therefore, it was able to be shown in animal experiments that non-infections synovial inflammation in arthritis models can be imaged using intravenously injected USPIO [173].

In a similar manner, the macrophage presence [174] and the increased extravasation of USPIO [175] in the edge region of bacterial soft tissue abscesses were able to be shown in T2w and T2*w MRI after i. v. USPIO application.

Moreover, macrophages were able to be detected experimentally as markers of low-grade chronically inflamed activity of fat tissue in adipose mice after the i. v. injection of USPIO [176].

Imaging of multiple sclerosis (MS) and neurodegeneration

When using SPIO to image the CNS, neuroinflammation (MS) and neurodegeneration (e. g. Alzheimer plaques) are in the foreground. In small animal models of MS (experimental autoimmune encephalomyelitis (EAE)), SPIO can be used to image macrophage activity [177] and infiltration [178], lymphocyte infiltration (CD3⁺ cells [179]) and the labeling of T-cells [180]. Also in the case of heart attack, numerous studies show that SPIO can image the accompanying neuroinflammation or the macrophage activity [181] and macrophage migration [182] similarly to the changes in MS. Moreover, it was able to be shown that early stages of post-radiogenic brain damage can be imaged via targeted SPIO-AB against ICAM-1 [183].

With respect to the use of SPIO in the imaging of neurodegenerative diseases such as Alzheimer’s disease, amyloid beta plaques were able to be successfully addressed via MION-AB [184] or SPIO-AB against amyloid-beta 42 [185].

Metabolic imaging

Newer studies show the promising use of SPIO in lipid-metabolism MR imaging by embedding in lipoprotein carriers, such as nanosomes [25]. In these studies the enzyme activity of the lipoprotein lipase in brown adipose tissue in rodents was able to be visualized via SPIO nanosomes [26]. Like ¹⁸FDG-PET, this type of imaging using SPIO nanosomes therefore has the potential to image metabolic conditions, i. e. enzymatic activity in tissues, in real time (Fig. [ 7 ]).

Fig. 7 Metabolic imaging. a Interscapular brown adipose tissue (BAT) of mice exposed to cold before (upper left) and after (upper right) i. v. application of SPIO-labeled triglyceride-rich lipoproteins (SPIO-TRL) with significant signal loss of the BAT due to cellular uptake of the SPIO-TRL (white arrow tips). b–d TEM images prove the internalization of SPIO-TRL in brown adipocytes and aggregation in endosomes (scale bar: b 1 µm, c 0.05 µm, d 20 nm; from Bartelt et al. [26]).

Imaging of enzymatic activity

In addition to the use of nanosomes, other studies describe the successful visualization of enzymatic activity in tissues in MRI using so-called activatable “smart sensor probes” [186] [187] [188]. These probes are initially inactive but generate a significant contrast in the presence of the target enzyme due to a change or switch in their magnetic relaxivity (magnetic relaxivity switch (MRS)) as a result of a transition from a disperse state to an aggregated state [188] [189] [190]. The principle of MRS could be shown both for specially prepared T1 contrast agents (e. g. β-galactosidase activity [191]) and for VSOP or CLIO (e. g. metalloproteinase-9 activity [192], telomerase activity [193] [194]) and can be used for in vitro MRS assays as well as potentially in vivo. For example, MMP-9-activatable protease-specific VSOP (MMP9-PSOP) lose their stabilizing coating of PEG copolymers in the presence of the target enzyme (protease) due to the enzyme-specific splitting of a coating-binding protein. As a result of the consequently accessible positive (coupling proteins) and negative charges (citrate coating) of the VSOP coating, particle aggregation, R2 relaxivity increase, and focal reduction of the T2w and T2w* MR signal occur and can be used as a measure of the enzyme/protease activity. The VSOP-based protease-sensitive nanosensors (PSOP) introduced by Schellenberger et al. have a number of properties favoring potential in vivo use as a “reporter probe” in MRI since these can be synthesized comparatively easily and can in principle be used for different proteases, and the particle size of PSOP (~25 nm) in association with the mPEG coating guarantees long bioavailability [192]. In principle, the MRS method should also be able to be transferred to other proteolytic enzymes (nucleases, polysaccharidases, proteases), which can be used in in vitro assays and potentially also in in vivo MR diagnostics [190].

Comparison of SPIO and Gd-containing contrast agents (Gd-CA) in MRI

Although problems with the use of SPIO have been reported in individual cases (e. g. allergic reactions), these are generally to be viewed as a safe contrast agent [63] [195] [196] [197] [198] [199] [200] [201]. Due to the relatively low molecular size of Gd-CA (about 1 nm), the simple application properties, the good tissue penetration and extravasation (dynamic contrast agent properties), the fast renal elimination and resulting ability to use them multiple times in short intervals, and the less artifact-prone imaging, Gd-CA are the contrast agent of choice in many areas of application and SPIO remain a supplementary contrast agent. Exceptions include patients with limited kidney function or renal insufficiency in whom SPIO can be a safe alternative to prevent contrast-induced nephropathy or nephrogenic systemic fibrosis [71]. A disadvantage of currently used, often polydisperse, SPIO and SPIO conjugates is the frequently short blood circulation time and thus the short target structure contact time due to the fast uptake in the liver, spleen, bone marrow, which often results in an only minimal accumulation in the target structure and tracer dispersion in non-targeted tissue. The advantage of the higher sensitivity of MRI for SPIO (mmol-µmol) can typically only conditionally compensate for this disadvantage. Nonetheless, cellular or molecular MR imaging and therapy are currently a domain of SPIO [29]. Newer synthesis methods producing monodisperse SPIO with better defined physicochemical and pharmacodynamic properties could greatly increase the importance of SPIO-enhanced MRI in the future [202] and the frequency of SPIO use in contrast-enhanced MRI.

Molecular therapy

In principle, the treatment of tumors using targeted SPIO can follow three different approaches: 1) Targeted SPIO bind specifically to the tumor receptor which selectively suppresses tumor growth, 2) targeted SPIO are used for magnetically induced hyperthermia after tumor binding, and 3) targeted SPIO are loaded with therapeutic agents (drug targeting) and these accumulate in the target tissue [23].

The effective binding and tumor growth inhibition of SPIO-anti-Her2-AB against breast cancer cells (SKBr3) [203], of anti-EGFRvIII-SPIO against glioblastoma cells [204], or of cetuximab-anti-EGFR immunomicelles against A431 malignant cells [205] was shown in the first approach.

The second strategy was able to be successfully shown by binding magnetoliposome-antibody complexes to MN antigens of renal cell carcinoma cells [206], magnetoliposome-anti-Her2-AB to breast cancer cells [203], or anti-folate receptor-SPIO to different tumor cell lines [207].

The third strategy was able to be successfully proven on the basis of rhenium188-loaded immuno(hepama-1)magnetic nanoparticles (Re-SPIO-AB) against liver carcinoma cells [208], methotrexate-conjugated SPIO against breast (MCF7) and cervical (HeLa) carcinoma cells [209], doxorubicin-loaded nanomicelles [210], doxorubicin-carrying hyaluronan-SPIO (DOX-HA-SPIO) [211], docetaxel-carboxymethylcellulose-SPIO [212], layer-by-layer (LbL) polyelectrolyte capsules that can be loaded with SPIO and/or therapeutic agents [213], SPIO- and doxorubicin-loaded cetuximab-anti-EGFR immunomicelles or with doxorubicin(liposomal)-loaded macrophages (macrophage-LP-Dox) that accumulate in tumors [214].

Although major advances in tumor labeling and tumor therapy via therapeutic imaging SPIO constructs (theranostics) have been made in recent years, the development of potent in vivo theranostics with high specificity and sensitivity has remained a significant challenge due to the heterogeneity of the expression level of the target structure on the tumor cells and problems with overcoming physiological barriers (e. g. extravasation or blood-tissue barriers) preventing access to the target cell (pharmacological accessibility).

Magnetic beads, MACS, and magnetofection

In addition to their use in imaging, particularly MRI, and in molecular therapy (hyperthermia, therapeutic agent transporter, or gene transporter), SPIO are also used in other biomedical applications. With the use of SPIO in in vitro diagnostics since the 1970 s [5], the spectrum has also continually expanded in this regard. Using magnetic beads in cytobiological research, this includes the in vitro filtration and sorting of proteins and peptides, nucleic acids, and cells (e. g. tumor or stem cells) [215] [216] and in vitro magnetofection of cells with DNA [217].

Iron oxide particles with a size of approximately 50 nanometers to several micrometers (microbeads) are referred to as magnetic beads. They have good magnetic properties and often a coating of silicon oxide or polystyrene and different coupling groups, such as streptavidin, carboxyl groups, protein A or G [218] [219]. In addition to iron oxide particles (γFe₂O₃/Fe₃O₄), cobalt, manganese, and nickel ferrite particles (CoFe₂O₄, MnFe₂O₄, NiFe₂O₄) are also used. These beads are then conjugated with a specific antibody for the target ligand [220].

During magnetic activated cell sorting (MACS), specific binding to the target cell structure occurs, these structures flow through a magnetic column and the targeted cells remain in the column. After washing to eliminate the non-magnetically bound cells, the selected, magnetically bound cells can be acquired in another step by removing the magnetic field [216].

During magnetofection (MF), nanoparticle-associated vector DNA is transfected by an external magnetic field to cells [217] [221]. The association of the negatively charged DNA is achieved by a positive surface charge of the nanoparticles via polycationic polyethylenimine. It was able to be shown that the effectiveness of the vector transfection can be increased several thousand times with only a minimal increase in toxicity [217] [222].

Diagnostic magnetic resonance (DMR)

Further applications of magnetic nanoparticles (MNP) with a high potential for further clinical diagnostics describe the in vitro use of targeted MNP or SPIO in quantitative diagnostic magnetic resonance (DMR) for the highly sensitive detection of biomarkers [223] (DNA, mRNA, proteins, small molecules, enzymes, medications), pathogens (viruses [224], bacteria [225]), or cells (tumor cells [226]), circulating immune/tumor cells [227]) under modulation of the T2 relaxation of biological samples.

Using miniaturized, chip-based DMR detector systems, it was able to be shown that highly sensitive, multiple DMR measurements can be performed using sample volumes of only several microliters (< 10 µl) [223]. Using portable micro-NMR (µNMR) [225] [227], it was able to be shown that this new diagnostic bedside technology can be used similarly to the principle of immunohistochemistry to analyze cell samples from tumors directly after acquisition (e. g. fine-needle aspiration) via specific, CLIO-labeled antibodies for a defined number of surface proteins (e. g. EpCAM (epithelial cell adhesion molecule), MUC-1 (mucin 1), HER2, EGFR, B7–H3, CK18, Ki-67, p53, vimentin). However, in contrast to conventional immunohistochemistry, a cell analysis can be performed in significantly less time (15 – 60 min.) with this method. In a clinical study on 70 test subjects with abdominal carcinomas, Haun et al. [226] were able to show that this still experimental-preclinical method can detect tumor cells with an accuracy of 96 % using a 4-protein signature (MUC-1 +EGFR+HER2 +EpCAM), that protein signatures of tumor cells depend on the extraction site within the tumors (tumor heterogeneity), and the signature can change from the time of sampling to cytological analysis making it necessary to perform real-time analysis. In clinical application, this method could make it possible to perform real-time analyses of tumor or blood cells directly ex vivo after sampling so that therapeutic treatment strategies can be introduced more quickly and can be better tailored to the in vivo situation.

Advantages of MNP-based analysis methods versus conventional ones could be the low intrinsic (background) magnetization of biological samples, the highly sensitive measurability of MNPs, and the short sample preparation time. Moreover, compared to optical techniques, DMR is not subject to interference such as light scattering, absorption, auto-fluorescence, or time-consuming sample preparation steps [223].

Magnetic particle imaging (MPI)

A new imaging modality using SPIO is magnetic particle imaging (MPI) [27]. This new radiation-free tomographic imaging method provides background-free, directly quantifiable information about the spatial distribution of SPIO with a high temporal resolution (milliseconds), spatial resolution (< 1 mm), and sensitivity (µmol) [228]. Feasibility in living organisms was able to be shown in initial preclinical studies [229]. Moreover, with optimization of the SPIO (currently often Resovist^®) and the equipment hardware, this technology has the potential to image nanomolar and picomolar concentrations of SPIO [230], making the application thereof in molecular imaging interesting. Using the currently available SPIO, similar application areas as in MRI combined with higher spatial and temporal resolution and better sensitivity are possible for this method. Potential MPI application areas include cardiovascular applications (angiographies, cardiac vitality diagnosis, tissue perfusion, plaque labeling, endovascular interventions, bleeding source diagnosis) or applications in tumor, molecular, and cellular imaging (passive and active targeting, molecular therapies, cellular labeling and cell monitoring) [231]. The advantages and disadvantages of this imaging method and possible clinical application areas will soon be demonstrated by an initiative of the German Research Foundation to install MPI small animal scanners and evaluate them in various disease entities.

Conclusion and outlook

Since MRI is an integral part of today’s clinical diagnosis of diseases, the search for suitable and targeted contrast agents has driven the development and use of SPIO. The major advances in materials research, nanotechnology, and conjugation chemistry have continuously expanded the application spectrum of SPIO in preclinical-experimental imaging in recent years. This is no longer limited to macroscopic-anatomical imaging using SPIO and is now also being applied to cellular and molecular imaging. The advantage of MRI in this regard is the simultaneous recording of anatomical, cellular, and molecular information. Due to the large surface and the chemically defined surface structure, SPIO can be conjugated with numerous molecules or functionalized (targeting molecules) and loaded with fluorescent dyes, radioisotopes, and therapeutic agents. The use of monodisperse SPIO with more precise physicochemical and pharmacodynamic characteristics could allow more exact addressing of target structures and more exact parametric or quantitative SPIO-MRI. The thus synthesized nanoparticles have the potential to revolutionize medical treatment and therapy in the coming years with improved early detection, multimodal imaging, and synchronous treatment and diagnostics (theranostics). Moreover, highly sensitive MR nanoprobes can be helpful in cell therapy concepts such as the transplantation of stem cells, islet cells, or immune cells since SPIO allow tracking of very small cell quantities in an organism. The use of highly specific MNP probes in MACS for magnetofection or in particular in chip-based µNMRs in diagnostic magnetic resonance (DMR) for the analysis of biological (patient) samples (e. g. biopsies) with respect to biomarkers, pathogens, or (tumor) cells could decisively expand the conventional method spectrum with respect to cell analysis (FACS, immunohistochemistry). MPI as a new imaging method currently being tested has the potential to open up a completely new spectrum in the application of SPIO in the case of successful evaluation in preclinical disease models.

It is currently still unclear whether the numerous SPIO constructs/conjugates successfully tested in preclinical models can fulfill the requirements of clinical application in the future and can prove their additional (compared to T1 contrast agent) or special benefit in MRI, DMR or MPI and this will remain the subject of research for the coming years and decades. In particular, questions regarding the safety profile of SPIO and SPIO conjugates, their pharmacokinetics and long-term toxicity, and the core as well as coating materials must be answered to ensure safe application of these nanoparticles in humans.

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