Ferric chloride (FeCl3, >97%), ferrous chloride tetrahydrate (FeCl2·4H2O >99%), riboflavin-5′-monophosphate sodium salt dihydrate (flavin mononucleotide; FMN, 73%–79% fluorometric), IDRANAL III® standard solution (EDTA-Na2, reagent for metal titration, 0.1 M), 5-sulfosalicylic acid dihydrate (≥99% for metal titration), 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate (Tiron®) indicator, and trypan blue solution for microscopy were obtained from Sigma Aldrich GmbH (Steinheim, Germany). Ammonia (NH3 ≥ 25%), N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES, ≥99.5%), hydrochloric acid (HCl, 37%), K4[Fe(CN)6], nuclear fast red, and hematoxylin and eosin were purchased from Carl Roth GmbH (Karlsruhe, Germany). Guanosine-5′-monophosphate disodium salt hydrate (GMP, 98%), gelatin (ph. Eur. powder), phosphate-buffered saline (PBS) (10× Dulbecco’s powder) were procured from Applichem (Darmstadt, Germany). Feraspin XS® used in this study was provided as a gift sample by Miltenyi Biotech GmbH (Bergisch Gladbach, Germany). Feraheme® was obtained from AMAG Pharmaceuticals, Inc. (Waltham, MA, USA). All chemicals were used as received without any further purification.
Synthesis of iron oxide nanoparticles (Fe3O4)
USPIO nanoparticles were synthesized by coprecipitation of ferrous (Fe2+) and ferric (Fe3+) salts under aqueous alkaline conditions as previously described [17, 18]. Briefly, in a stoichiometric ratio of 2Fe3+:Fe2+, 16 mmol (2.66 g) FeCl3 and 8 mmol (1.63 g) FeCl2·4H2O dissolved in 190 mL de-ionized water were co-precipitated using 10 mL 25% NH3. The nanoparticles were washed with water and 0.1 M HCl and stored under acidic conditions (pH 2) until further use. The total iron content (Fe3+ ions) of USPIO (161 mM) was determined by titrimetric  and colorimetric  methods.
Synthesis of FLUSPIO nanoparticles
FLUSPIO nanoparticles were prepared as previously described . Briefly, 143 mM USPIO (pH 4) was sonicated with 35 mM FMN for 1 h at ambient temperature. Subsequently, the FMN-covered nanoparticles were washed with water under the influence of highgradient magnetophoresis and then sonicated with 50 mM GMP for 1 h. After final coating and highgradient magnetophoresis-assisted washing with water, the particles were stored at 4 °C until further use.
Atomic force microscopy (AFM)
Samples for AFM analysis were prepared using FLUSPIO (5 μL) in water, which were deposited onto a polymer membrane (Nucleopore 0.1 μm, Whatman) that provided a flat surface to visualize the nanoparticles. Samples were dried in a fume hood for 1.5 h, which led to the formation of a ring of dried material at the perimeter of the dried droplet, providing a high concentration of particle aggregates as a target for AFM imaging.
All AFM images were captured in tapping mode on a multimode nanoscope IIIa instrument (Veeco, USA) using BudgetSensors Tap300 probes. Topographic and phase images were recorded simultaneously from the same area of sample.
Dynamic light scattering (DLS) measurements
The size distribution of USPIO and FLUSPIO in 10 mM HEPES buffer (pH 7.2) with different concentrations of human serum albumin (HSA) was investigated by DLS using the Malvern Zetasizer, Nano-S (Malvern, UK).
Zeta potential measurements
Zeta potential and electrophoretic mobility of USPIO and FLUSPIO were determined using a Malvern 4700 system (Malvern Ltd., Malvern, UK). Freshly prepared nanoparticles (0.5 mL) were diluted (1,000-fold) in water, 5% (w/v) glucose solution, fetal calf serum (FCS), 1:1 (v/v) mixture of 5% glucose solution and FCS, or 25 mM HEPES buffer (pH 7.2), respectively. After dilution, each sample was measured on an average of seven runs (± the standard deviation) in triplicates. Intralipid was used as a reference for zeta potential measurements.
The fluorescence spectra of FMN and FLUSPIO suspended in different physiologically relevant solutions (5% (w/v) glucose solution, FCS alone, and 1:1 (v/v) mixture of 5% glucose solution and FCS) were recorded for different fluorophore (FMN) and iron (FLUSPIO) concentrations ranging from 10–5 to 10–12 M (37 °C) using the TECAN Infinite M200pro (Tecan group, Maennedorf, Switzerland) plate reader.
Magnetic resonance (MR) relaxometry (phantom studies)
MR relaxometry of FLUSPIO and Feraspin XS® (Miltenyi Biotech, Germany), a preclinical MR contrast agent for visualizing vasculature, was performed using a clinical (3T) whole-body MR scanner (Philips Achieva, The Netherlands) in combination with a knee coil (SENSE-flex-M, Philips, The Netherlands) at room temperature. FLUSPIO and Feraspin XS® were diluted in de-ionized water at concentrations ranging from 0.005 to 50 μg Fe/mL. For MR measurements, 0.3 mL diluted FLUSPIO and Feraspin XS® solution was filled in custom-made phantoms. For T
2 relaxometry in phantoms, images were acquired at 20 echo times (TE range = 8–180 ms) using a multi-slice, multi-shot, spin-echo sequence (1,500 ms TR, 8.1 ms inter-echo spacing, 96 × 96 reconstruction matrix size, 2 mm × 2 mm voxel size, 3-mm slice thickness, 130 mm × 162.5 mm field of view, 90° flip angle). Regions of interest (ROI) were manually defined. T
2 relaxation times were calculated by a linear fit of the logarithmic signal amplitudes versus echo time.
1 maps were acquired using a multi-slice, multishot, fast-field-echo Look-Locker inversion recovery sequence using simulated electrocardiogram triggering (30 bpm stimulated heart rate, 70-ms phase interval, 6–8 ms TR, 3–4 ms TE, 160 × 160 reconstruction matrix, 5-mm slice thickness, 170 mm × 150 mm field of view, 10° flip angle). Relaxation signal amplitudes were fitted to a signal model to yield T
1 relaxation times.
Relaxivities r1 and r2 for FLUSPIO and Feraspin XS® were determined by a linear fit of the inverse relaxation times as a function of the iron concentrations.
Time-of-flight secondary ion mass spectrometry (TOF-SIMS)
TOF-SIMS is a high-vacuum surface technique, which provides details about a surface (1–2 nm analysis depth) with high chemical sensitivity (ppm). The surface chemical composition of USPIO and FLUSPIO samples was determined using the Ion ToF IV instrument (ION-TOF GmbH, Münster, Germany). Samples were prepared as described in the AFM section 2.4.1 above.
X-ray photoelectron spectroscopy (XPS)
The quantitative surface elemental composition of USPIO and FLUSPIO samples was determined using a Kratos Axis Ultra XPS instrument (Kratos Axis Ultra, Manchester UK). This technique provides quantitative information from a surface analysis depth of 5–10 nm.
Stability of FLUSPIO in physiological solutions
The colloidal stability of FLUSPIO suspended in different physiologically relevant solutions was checked at different settling times. For our study, we suspended 3.0 μmol Fe/mL FLUSPIO in water, 5% (w/v) glucose solution, FCS alone, 1:1 (v/v) mixture of 5% glucose solution and FCS, and 25 mM HEPES buffer (pH 7.2), respectively. Subsequently, photographs of the colloidal suspensions were taken at different time points (0, 0.5, 1, 3, 12, and 24 h) using a high-resolution digital camera.
LnCap cells were procured from American Type Culture Collection (ATCC), USA. Human umbilical vein endothelial cells (HUVEC) were obtained from Promocell GmbH, Heidelberg, Germany. LnCap cells were cultured in RPMI medium with Glutamax (Gibco, Invitrogen, Germany), 20% FCS (Invitrogen, Germany) and 1% Pen/Strep (10,000 U/mL penicillin; 10,000 μg/mL streptomycin, Invitrogen, Germany). HUVEC were grown in human endothelial cell growth medium (VascuLife VEGF, Lifeline Cell Technology, Troisdorf Germany) containing growth supplements, 2% FCS and 1% Pen/Strep. Cells were cultured in T75 cm2 cell culture flasks (Cell Star, Greiner, Germany) and incubated at 37 °C under 5% CO2 and 95% relative humidity conditions until they were confluent.
Cellular viability testing with trypan blue staining
The viability of LnCap cells and HUVEC after incubation with FLUSPIO was evaluated using trypan blue staining (trypan blue solution, Sigma-Aldrich, Steinheim, Germany). Cells (2 × 106) were seeded in 6-well plates (BD Falcon, Germany) and incubated overnight. After incubation, medium was removed and cells were washed with PBS. Cells incubated with cell growth media served as negative controls. The positive controls were generated by exposing the cells to 90 °C for 10 min. Further control groups were maintained under normal growth conditions but after adding the coating molecules FMN and GMP to the growth medium at different concentrations. USPIO and FLUSPIO were diluted in respective cell growth media to 0.03, 0.3, 3.0 μmol Fe/mL and cells were incubated for 3 and 24 h, respectively. Triplicates per condition were analyzed. Subsequently, cells were washed, trypsinized (0.25% trypsin/0.05% EDTA) and centrifuged. Equal amounts of the cell suspension and trypan blue solution were mixed prior to measurement. Trypan blue-positive cells (dead cells) were counted using a Cedex XS cell counter and the percentage of positive cells was reported as a function of the total cell count.
Competitive binding studies (Prussian blue staining)
To evaluate the specific binding of FLUSPIO to LnCap cells and HUVEC, competitive binding studies were performed. Cells were seeded on glass slides (76 mm × 26 mm) placed inside a quadriPERM chamber (Greiner, Germany) and incubated overnight to allow them to adhere onto the surface. Prussian blue iron staining was used to visualize FLUSPIO (0.3 μmol Fe/mL) uptake and its inhibition after addition of 10- or 100-fold excess of free FMN for 1 h. Additionally, controls were carried along where cells were incubated with cell growth medium only or with USPIO (0.3 μmol/mL). After incubation for 1 or 3 h, cells were washed thrice with PBS and fixed using 4% paraformaldehyde solution. Subsequently, the fixed cells were treated for 5 min with an aqueous solution of K4Fe(CN)6 (10%) and then with a 1:1 mixture of K4Fe(CN)6 (10%) and HCl acid (20%) for 30 min. Subsequently, cells were counterstained with Nuclear Fast Red (Carl Roth, Karlsruhe, Germany). Cover slips were mounted using Mowiol medium and cells were analyzed by brightfield microscopy (Imager M2, Carl Zeiss Microimaging GmbH, Germany) at different magnifications and fields of view.
MR characterization of competitive binding studies
Specificity of FLUSPIO nanoparticles for Rf receptors expressed in LnCap cells and HUVEC was studied by MRI through competitive-binding studies. Cells were pre-incubated for 10 min with 10-fold excess of free FMN. Consecutively, FLUSPIO (0.3 μmol/mL) were added and cells were incubated for 1 h at 37 °C. Cell growth medium, USPIO (0.3 μmol/mL) and Feraheme® (0.3 μmol/mL) were used in control samples under comparable conditions. Three samples were analyzed per condition. After incubation, cells were washed with saline solution and trypsinized by adding 4 mL trypsin/EDTA (0.25%/0.05%). Trypsinization was stopped by adding respective cell growth media and the cell suspension was centrifuged at 1,000 rpm (Multifuge, Thermo scientific, Germany) for 5 min. The pellet was washed thrice by centrifugation with saline. Cells (0.5 × 106 cells/0.3 mL) were suspended in 10% gelatin and analyzed by MR-relaxometry as described in section 2.4.5.
MR imaging of FLUSPIO accumulation in LnCap xenografts in mice
All animal experiments were approved by the government review committee on animal care. In this study, LnCap tumor xenografts were chosen since LnCap cells display high expression of RCP . LnCap tumor xenografts were induced by subcutaneous injection of 5 × 106 LnCap cells  into the right hind limb of 10 BALB/c male nude mice (Charles River Laboratories International, Inc., Wilmington, MA, USA). MR investigations were carried out when tumors reached the size of 4 mm × 4 mm (volume of approximately 33 mm3). The mice (n = 5) were then, intravenously injected with 900 μmol Fe/kg FLUSPIO. In the competitive-binding group (n = 5), mice were injected with 10-fold excess FMN at 10 min before FLUSPIO (900 μmol Fe/kg) administration. MR imaging of LnCap tumor xenografts pre- and post-particle injection (1 and 3 h) was performed under isoflurane anesthesia (2%) using a clinical 3T MRI system (Philips Achieva 3.0 T), in combination with a custom-built small animal solenoid sense-receive mouse coil .
To visualize anatomic details, transverse T
2-weighted MR images of the tumors were acquired using a multi-shot turbo-spin-echo sequence (1,400 ms/100 ms (TR/TE), 0.2 mm × 0.2 mm voxel size, 1-mm slice thickness, 144 × 144 reconstruction matrix size, and 25-mm field of view). T
-weighted MR images were acquired using a fast-field-echo sequence (117 ms/26 ms (TR/TE), 0.2 mm × 0.2 mm voxel size, 1-mm slice thickness, 96 × 96 reconstruction matrix size, 25-mm field of view and 30° flip angle). Subsequently, T
relaxometry was performed at the same location using a multi-shot, multi-slice fast-field-echo sequence (125 ms/(10–190 ms) TR/TE (TE with 15 different echo times), 0.3 mm × 0.3 mm voxel size, 1-mm slice thickness, 144 × 144 reconstruction matrix size, 40-mm field of view, 30° flip angle and 7.9-ms interval between two echoes).
Post-processing was performed on the basis of PAR/REC4 images by applying an IDL (version 6.1)-based software. Subsequently, color-coded R
relaxation rate pixel maps of tumors were generated using the PRIDE analysis software (Philips Healthcare, Germany).
Immunohistochemistry and bio-distribution
Immunofluorescence of LnCap tumors
LnCap tumor sections (8-μm thickness) were stained for endothelial cells (vessels) by incubation with a primary rat anti-mouse CD31 (PECAM-1) antibody (BD Bioscience, Heidelberg, Germany) for 2 h (room temperature), followed by Cy3-labeled anti-rat IgG secondary antibody (Dianova GmbH, Hamburg, Germany) for 45 min (room temperature). Subsequently, different LnCap tumor sections were stained for macrophages using a primary rat anti-mouse CD68 antibody (AbD Serotec, Duesseldorf, Germany) for 2 h (room temperature) combined with a Cy3-labeled anti-rat IgG secondary antibody for 45 min (room temperature). Nuclei in the tumor sections were counterstained with DAPI (Merck KGaA, Darmstadt, Germany). Fluorescence microscopy analysis (Imager M2, Carl Zeiss Microimaging GmbH, Germany) was performed at different magnifications (10×, 20×, and 40×) and fields of view.
Combined Prussian blue and hematoxylin-eosin (HE) staining of LnCap tumors
Cryo-preserved (–80 °C) LnCap tumors covered with Tissue-Tek (O.C.T. Compound, Sakura Finetek Europe B.V., Germany) were sectioned and then subjected to combined Prussian blue and HE staining. The Prussian blue staining protocol was performed as described in section 2.6, except that no nuclear fast red staining was performed. Subsequently, the tumor sections were treated with hematoxylin (Carl Roth GmbH, Germany) and with a 1% aqueous solution of eosin (Carl Roth GmbH, Germany). Then, tumor sections were washed with PBS, dehydrated by incubation with an increasing percentage of alcohol (70%, 96%, and 100%) and incubated in xylene. Cover slips were mounted on the sections using Vitro-Clud (R. Langenbrinck, Emmendingen, Germany), a xylenebased mounting medium before microscopic analysis (Imager M2, Carl Zeiss Microimaging GmbH, Germany).
Bio-distribution of FLUSPIO in tumor-bearing mice
For mice (n = 5) injected with FLUSPIO, after MRI measurement, liver, spleen, lung, kidney, heart, colon, bladder, muscle, and skin as well as LnCap tumors were excised and subjected to bio-distribution evaluation using an iron-based colorimetric assay. The excised organs and tumors were weighed prior to dissolving them in 3 mL aqua regia by sonication. Subsequently, 1 mL sample solution was diluted in water (1:2 and 1:10) and mixed with phosphate buffer, 4 M NaOH, and 0.25 M Tiron®. The absorbance of the iron-tiron complex was measured at 520 nm. Particlerelated iron uptake was determined by normalizing the estimated iron contents to organ weights and by subtracting the intrinsic iron content in organs/tumors derived from mice that were not administered FLUSPIO injections.
The differences between groups were tested for statistical significance using a two-tailed, unpaired student’s t-test considering p-values below 0.05 as significant.