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
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 r2* relaxivity of SPIO as the reciprocal of the T2 or T2* relaxation (r2 = 1/T2 or r2* = 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 r2* than for SPIO with a low r2 and r2*. 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(Fe3 +)magnetic and ferro(Fe2 +)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
(Fe2 + 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
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
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
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], ανβ3-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-Fab (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.
