and Alkoxysilane-Coated Ultrasmall Superparamagnetic Iron Oxide

Langmuir 2007, 23, 1427-1434

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Silica- and Alkoxysilane-Coated Ultrasmall Superparamagnetic Iron Oxide Particles: A Promising Tool To Label Cells for Magnetic Resonance Imaging Chunfu Zhang,†,‡ Bjo¨rn Wa¨ngler,†,‡ Bernd Morgenstern,†,‡ Hanswalter Zentgraf,§ Michael Eisenhut,| Harald Untenecker,⊥ Ralf Kru¨ger,∇ Ralf Huss,O Christian Seliger,O Wolfhard Semmler,‡ and Fabian Kiessling*,† Junior Group Molecular Imaging and Departments of Medical Physics in Radiology, of Applied Tumor Virology, of Radiopharmaceutical Chemistry, and of Spectroscopy, German Cancer Research Center, Heidelberg, Merck Company, Darmstadt, and Institute of Pathology, UniVersity of Munich, Munich, Germany ReceiVed June 29, 2006. In Final Form: October 19, 2006 In this study silica- and alkoxysilane-coated ultrasmall superparamagnetic iron oxide (USPIO) particles were synthesized, and their ability to label immortalized progenitor cells for magnetic resonance imaging (MRI) was compared. USPIO particles were synthesized by coprecipitation of ferric and ferrous salts. Subsequently, the particles were coated with silica, (3-aminopropyl)trimethoxysilane (APTMS), and [N-(2-aminoethyl)-3-aminopropyl]trimethoxysilane (AEAPTMS). The size of the USPIO particles was about 10 nm without a significant increase in diameter after coating. The highest T2 relaxivity was achieved for silica-coated USPIO particles, 339.80 ( 0.22 s-1 mM-1, as compared with APTMS- and AEAPTMS-coated ones, reaching 134.40 ( 0.01 and 84.79 ( 0.02 s-1 mM-1, respectively. No toxic effects on the cells could be detected by trypan blue, TUNEL, and MTS assays. Uptake of USPIO particles was evaluated by Prussian blue staining, transmission electron microscopy, T2-MR relaxometry, and mass spectrometry. It was found that cell uptake of the different USPIO particles increased for longer incubation times and higher doses. Maximum cellular iron concentrations of 42.1 ( 4.0 pg/cell (silica-coated USPIO particles), 37.1 ( 3.5 pg/cell (APTMS-coated USPIO particles), and 32.7 ( 4.0 pg/cell (AEAPTMS-coated USPIO particles) were achieved after incubation of the cells with USPIO particles at a dose of 3 µmol/mL for 6 h. The decrease of the T2 relaxation time of the cell pellets was most pronounced for cells incubated with silica-coated USPIO particles followed by APTMS- and AEAPTMS-coated particles, respectively. In gelatin gels even small clusters of labeled cells were detected by 1.5 T MRI, and significant changes in the T2 relaxation times of the gels were determined for 10000 labeled cells/mL for all particles. In summary, as compared with APTMS- and AEAPTMS-coated particles, silica-coated USPIO particles provide the highest T2 relaxivity and most effectively reduce the T2 relaxation time of immortalized progenitor cells after internalization. This suggests silica-coated USPIO particles are most suited for cell labeling approaches in MRI.

Introduction During the past several years there has been an increasing interest in developing tools for cellular and targeted magnetic resonance imaging (MRI). While MRI provides high spatial resolution and an excellent soft tissue contrast, its low sensitivity to contrast agents as compared with that of nuclear medicine techniques makes molecular MRI approaches often fail to visualize the specific targets in vivo. One way to overcome this problem is the use of MR scanners with higher magnetic fields. However, since the signal-to-noise ratio increases linearly with the field strength, only a moderate improvement in the sensitivity can be achieved. Even more evident than increasing the strength of the magnetic field is the design of appropriate MR contrast agents. In the clinical routine nonspecific low molecular weight * To whom correspondence should be addressed. Phone: +49 6221 422533; fax: +49 6221 422572; e-mail: f. [email protected]. † Junior Group Molecular Imaging, German Cancer Research Center. ‡ Department of Medical Physics in Radiology, German Cancer Research Center. § Department of Applied Tumor Virology, German Cancer Research Center. | Department of Radiopharmaceutical Chemistry, German Cancer Research Center. ⊥ Merck Co. ∇ Department of Spectroscopy, German Cancer Research Center. O University of Munich.

gadolinium chelates are almost exclusively used as MR contrast agents that decrease the T1 relaxation time of tissue due to interaction with surrounding protons. However, high local amounts of gadolinium are required to successfully visualize cells or specific targets. Therefore, for specific or cellular MR labeling gadolinium-containing liposomes are used.1,2 However, the latter approaches are all in an in vitro or early experimental in vivo state. Contrast agents consisting of (ultrasmall) superparamagnetic iron oxide ((U)SPIO) particles are an alternative that generate a signal reduction in T2- and T2*-weighted images. With the exception of very small superparamagnetic iron oxide particles with dedicated coatings,3 the ratio between T1 and T2 relaxivity is usually low, which results in a low T1 contrast. Some SPIO and USPIO particles coated with dextran and its derivatives, such as Feridex (Endorem) and Resovist, are commercially available and clinically used for the characterization of liver lesions4 and lymph nodes.5 Both applications are based on the (1) Mulder, W. J.; Strijkers, G. J.; Habets, J. W.; Bleeker, E. J. van der Schaft, D. W.; Storm, G.; Koning, G. A.; Griffioen, A. W.; Nicolay, K. FASEB J. 2005, 19, 2008-2010. (2) Daldrup-Link, H. E.; Rudelius, M.; Oostendorp, R. A.; Settles, M.; Piontek, G.; Metz, S.; Rosenbrock, H.; Keller, U.; Heinzmann, U.; Rummeny, E. J.; Schlegel, J.; Link, T. M. Radiology 2003, 228, 760-767. (3) Taupitz, M.; Schnorr, J.; Abramjuk, C.; Wagner, S.; Pilgrimm, H.; Hu¨nigen, H.; Hamm, B. J. Magn. Reson. Imaging 2000, 12, 905-911. (4) Reimer, P.; Tombach, B. Eur. Radiol. 1998, 8, 1198-1204.

10.1021/la061879k CCC: $37.00 © 2007 American Chemical Society Published on Web 11/30/2006

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particle uptake by macrophages and thus the generation of a signal reduction in the non-neoplastic tissue. The labeling of cells in vitro using (U)SPIO particles or other MR contrast agents and their retransfer into the organism in combination with the noninvasive assessment of their distribution and migration in vivo is defined as “cell tracking”. Cell tracking has become a valuable tool in preclinical research on various diseases, e.g., for monitoring the accumulation of T lymphocytes in tumors,6 following the migration of dendritic cells to lymph nodes,7 and assessing the accumulation of progenitor cells in brain lesions, cardiac infarctions, and tumors.8-11 However, it has been shown that the uptake of (U)SPIO particles by cells is highly dependent on the cell type and the size, surface charge, and coating of the particles.12 Thus, loading (U)SPIO particles with peptides that mediate the phagocytosis, such as HIV-Tat, or the use of transfection agents is often required to sufficiently label the cells.13,14 While several approaches were optimized for cell labeling that included the use of magnetodendrimers15 and anionic magnetic nanoparticles,16-18 it can be concluded that there is still a demand for particles that have a high T2 relaxivity, that are intensely internalized by cells, and that can easily be labeled with small molecules to enhance the cellular uptake. In this context, surface coating of (U)SPIO particles using silica and alkoxysilanes is promising.19 Silica and alkoxysilanes are among the most frequently used surface coating materials for inorganic nanoparticles, especially iron oxide particles.20-28 Silica has the advantage of being biocompatible and hydrophilic. It prevents the aggregation of particles in liquids and improves (5) Anzai, Y.; Blackwell, K. E.; Hirschowitz, S. L.; Rogers, J. W.; Sato, Y.; Yuh, W. T.; Runge, V. M.; Morris, M. R.; McLachlan, S. J.; Lufkin, R. B. Radiology 1994, 192, 709-715. (6) Kircher, M. F.; Allport, J. R.; Graves, E. E.; Love, V.; Josephson, L.; Lichtman, A. H.; Weissleder, R. Cancer Res. 2003, 63, 6838-6846. (7) Ahrens, E. T.; Feili-Hariri, M.; Xu, H.; Genove, G.; Morel, P. A. Magn. Reson. Med. 2003, 49, 1006-1013. (8) Ben-Hur, T.; Einstein, O.; Mizrachi-Kol, R.; Ben-Menachem, O.; Reinhartz, E.; Karussis, D.; Abramsky, O. Glia 2003, 41, 73-80. (9) Kraitchman, D. L.; Heldman, A. W.; Atalar, E.; Amado, L. C.; Martin, B. J.; Pittenger, M. F.; Hare, J. M.; Bulte, J. W. M. Circulation 2003, 107, 22902293. (10) Hung, S. C.; Deng, W. P.; Yang, W. K.; Liu, R. S.; Lee, C. C.; Su, T. C.; Lin, R. J.; Yang, D. M.; Chang, C. W.; Chen, W. H.; Wei, H. J.; Gelovani, J. G. Clin. Cancer Res. 2005, 11, 7749-7756. (11) Ehtesham, M.; Steven, C. B.; Thompson, R. C. Neurosurg. Focus 2005, 19, E5. (12) Modo, M.; Hoehn, M.; Bulte, J. W. M. Mol. Imaging 2005, 4, 143-164. (13) Zhao, M.; Kircher, M. F.; Josephson, L.; Weissleder, R. Bioconjugate Chem. 2002, 13, 840-844. (14) Frank, J. A.; Miller, B. R.; Arbab, A. S.; Zywicke, H. A.; Jordan, E. K.; Lewis, B. K.; Bryant, L. H., Jr.; Bulte, J. W. M. Radiology 2003, 228, 480-487. (15) Bulte, J. W.; Douglas, T.; Witwer, B.; Zhang, S. C.; Strable, E.; Lewis, B. K.; Zywicke, H.; Miller, B.; van Gelderen, P.; Moskowitz, B. M.; Duncan, I. D.; Frank, J. A. Nat. Biotechnol. 2001, 19, 1141-1147. (16) Billotey, C.; Wilhelm, C.; Devaud, M.; Bacri, J. C.; Bittoun, J.; Gazeau, F. Magn. Reson. Med. 2003, 49, 646-654. (17) Wilhelm, C.; Billotey, C.; Roger, J.; Pons, J. N.; Bacri, J. C.; Gazeau, F. Biomaterials 2003, 24, 1001-1011. (18) Fleige, G.; Seeberger, F.; Laux, D.; Kresse, M.; Taupitz, M.; Pilgrimm, H.; Zimmer, C. InVest. Radiol. 2002, 37, 482-488. (19) Coradin, T.; Lopez, P. J. ChemBioChem 2003, 4, 251-259. (20) Philipse, A. P.; van Bruggen, M. P.; Pathmamanoharan, C. Langmuir 1994, 10, 92-99. (21) Yang, H. H.; Zhang, S. Q.; Chen, X. L.; Zhuang, Z. X.; Xu, J. G.; Wang, X. R. Anal. Chem. 2004, 76, 1316-1321. (22) Bruce, I. J.; Sen, T. Langmuir 2005, 21, 7029-7035. (23) Kohler, N.;. Fryxell, G. E; Zhang, M. J. Am. Chem. Soc. 2004, 126, 7206-7211. (24) Mikhaylova, M.; Kim, D. K.; Berry, C. C.; Zagorodni, A.; Toprak, M.; Curits, A. S.; Muhammed, M. Chem. Mater. 2004, 16, 2344-2345. (25) Fan, H.; Chen, Z.; Brinker, C. J.; Clawson, J.; Alam, T. J. Am. Chem. Soc. 2005, 127, 13746-13747. (26) Donselaar, L. N.; Philipse, A. P.; Suurmond, J. Langmuir 1997, 13, 6018. (27) Liu, Q.; Xu, Z.; Finch, J. A.; Egerton, R. Chem. Mater. 1998, 10, 3936. (28) Correa-Duarte, M. A.; Giersig, M.; Kotov, N. A.; Liz-Marzan, L. M. Langmuir 1998, 14, 6430.

Zhang et al.

their chemical stability.26-28 Furthermore, the surface of silicacoated particles is often terminated by a silanol group that can react with various coupling agents to covalently attach specific ligands.29 Alkoxysilanes are bifunctional molecules with the general formula [X(CH2)n]mSiR(OR′)3-m, where X represents the headgroup functionality, (CH2)n a flexible spacer, and Si(OR)n the anchor groups by which they can attach to free SiOH or FeOH surface groups after hydrolysis of the alkoxy group.22 In contrast to silica coatings, which need further surface modification for coupling bioactive molecules, this can be done directly using alkoxysilanes with functional groups, such as amino groups. Silica- and alkoxysilane-coated iron oxide particles were used as carriers of enzymes to investigate biological catalysis,21 to perform magnetic bioseparation,22 and to label epithelial cells in vivo.30 There was also a report on the principle possibility to use silica-embedded iron oxide nanoparticles as a contrast agent in MRI.31 However, these particles had a large average diameter of about 220 nm and several particles in one encapsulation. Thus, the intention of this study was to synthesize and characterize USPIO particles with silica and alkoxysilane coatings and to evaluate their potential to label immortalized rat progenitor cells for cellular MRI. Materials and Methods Ferric chloride (FeCl3; >97%), ferrous chloride tetrahydrate (FeCl2‚4H2O; >99%), sodium hydroxide solution (1 M), sodium metasilicate pentahydrate (Na2O3Si‚5H2O), hydrochloric acid (HCl; 1 M solution), methanol (>99.8%), toluene (>99.8%), (3-aminopropyl)trimethoxysilane (APTMS; >97%), [N-(2-aminoethyl)-3aminopropyl]trimethoxysilane (AEAPTMS; >98%), nucleus fast red, and Prussian blue were purchased from Sigma-Aldrich (St. Louis, MO). Milli-Q purified water was used for all experiments. Synthesis and Characterization. Synthesis of USPIO Nanoparticles. For the preparation of USPIO nanoparticles, a modified Massart method was used.32 A mixture of FeCl3 (20 mL, 1 M in 0.2 M HCl) and FeCl2 (20 mL, 0.5 M in 0.2 M HCl) was added dropwise to a mechanically stirred solution of sodium hydroxide (200 mL, 1.5 M) within 5 min under an inert nitrogen atmosphere at 80 °C. The mixture was stirred for 20 min and subsequently cooled to ambient temperature. The sediment was separated by an external magnetic field, washed three times with deionized water, and dispersed into 100 mL of aqueous hydrochloric acid (0.01 M). Surface Modification of USPIO Particles with Silica, APTMS, and AEAPTMS. Using sodium metasilicate pentahydrate as the silica source, a thin layer of silica could be deposited onto the USPIO particles.20 Briefly, a solution of 200 mL of 0.58 wt % SiO2 (from Na2O3Si‚5H2O) was passed through a Dowex 50 W×4 acid ion exchange column which was previously regenerated by subsequent flushing with hot doubly distilled water (about 100 °C) and 3 M HCl and cooled to ambient temperature using doubly distilled water. The USPIO particles (1 g) were dispersed into the eluent after adjustment of the pH value to 9.5 with 0.5 M sodium hydroxide solution. The suspension was incubated at 80 °C for 2 h with vigorous mechanical stirring. The coated particles were separated with the aid of a magnet and washed three times with deionized water. Finally the particles were dispersed into water. A thin silane coating was achieved by a direct silanizing of the USPIO particles with APTMS.24 USPIO particles (1 g) were first washed with methanol (50 mL), thereafter with a mixture of methanol and toluene (50 mL, v/v ) 1:1), and finally with toluene alone (50 mL). Subsequently, the particles were dispersed into toluene (100 (29) Ulman, A. Chem. ReV. 1996, 96, 1533. (30) Dormer, K.; Seeney, C.; Lewelling, K.; Lian, G.; Gibson, D.; Johnson, M. Biomaterials 2005, 26, 2061-2072. (31) Yan, F.; Xu, H.; Anker, J.; Kopelman, R.; Ross, B.; Rehemtulla, A.; Reddy, R. J. Nanosci. Nanotechnol. 2004, 4, 72-76. (32) Massart R. Trans. Magn. 1981, 17 (2), 1247-1249.

Silica- and Alkoxysilane-Coated USPIO Particles mL), and APTMS (0.1 mL, 5.73 mol/L) was added dropwise to the suspension within 5 min. The ferrofluid suspension was transferred into a three-necked flask with N2 flow and refluxed under mechanical stirring for 10 h. The modified particles were magnetically collected and redispersed into methanol by ultrasonication. After being washed with methanol, the APTMS-coated USPIO particles were dispersed into water. The process for AEAPTMS coating was analogous, with the exception that AEAPTMS was added to the USPIO-toluene suspension to achieve a final concentration of 4.6 mM. X-ray Diffraction (XRD). The crystal structures of the naked USPIO particles and modified ones were characterized by XRD, which was carried out in a D/max 2550V diffractometer using the KR line of Cu (λ ) 1.5418 Å) as a radiation source. Transmission Electron Microscopy (TEM). The morphology, size, and size distribution of the particles were characterized by TEM (JEOL-100CX). USPIO particles were placed onto a glow-discharged carbon-coated 300 mesh copper grid for 1 min. After the grid was rinsed with water it was immediately air-dried. The particle size and size distributions were calculated using an image analysis program and by measuring the diameter of at least 300 particles. USPIO-labeled cells were grown on glass coverslips and fixed with ice cold 2.5% glutaraldehyde in 0.05 M sodium cacodylate (pH 7.2) for 20 min on ice. The slides were successively stained with 2% osmium tetroxide and 0.5% uranyl acetate and processed for ultrathin sectioning. Micrographs were taken with a Zeiss EM-10 A electron microscope at 80 kV. The magnification indicator was routinely controlled by the use of a grating replica. Surface Elementary Analysis of the Coated USPIO Particles. For USPIO particles with the alkoxysilane coating organic elementary analysis was carried out with inductively coupled plasma atomic emission spectrometry (ICP-AES) (IRIS Advantage 1000) and a Vario EL elementary analysis instrument (Elementar, Germany). The inorganic components were evaluated by an energy-dispersive X-ray (EDX) detector attached to the scanning electron microscope (LEO 1530 VP). Determination of the Surface Charge of Silica-, APTMS-, and AEAPTMS- Coated USPIO Particles. The surface charge of modified USPIO particles was determined by measuring the ζ potential as a function of the pH value of USPIO suspensions using a particle charge detector (PCD 03, Muetec, Herrsching, Germany). MR Relaxometry of USPIO Particles. Nuclear MR relaxometry of the particles was performed using a clinical 1.5 T whole-body MR system (Siemens Symphony, Erlangen, Germany) in combination with a custom-made radio frequency coil for excitation and signal reception. The radio frequency coil was designed as a cylindrical volume resonator with an inner diameter of 83 mm and a usable length of 120 mm. To optimize the available signal-to-noise ratio, manual tuning and matching of the coil’s resonance circuitry was performed for each measurement. USPIO particles were diluted in distilled water at concentrations between 0.001 and 100 µg of Fe/ mL. For MR measurements 0.5 mL USPIO dilutions were filled in 1 mL Eppendorf vials each. T2 relaxation times were measured using a standard Carr-Purcell-Meiboom-Gill pulse sequence (TR ) 2000 ms, TE range 30-960 ms, 32 echoes, FOV ) 134 × 67 mm, matrix 128 × 64, slice thickness 10 mm, BW ) 40, NEX ) 3). T2 relaxation times were calculated by a linear fit of the logarithmic ROI signal amplitudes versus TE. T1 relaxation times were determined using a T1w saturation recovery turbo FLASH sequence, thereby varying the inversion time, TI (TR ) 7160 ms, TE ) 1.67 ms, TI ) 90-4000 ms, FOV ) 200 × 150 mm, matrix 52 × 128, slice thickness 2 mm). The T1 and T2 relaxivities were determined by a linear fit of the inverse relaxation times as a function of the iron concentrations used. Cell Labeling. Experiments on USPIO uptake and particle toxicity were performed using immortalized rat progenitor cells, which were already used for similar experiments on dextran-coated SPIO and

Langmuir, Vol. 23, No. 3, 2007 1429 USPIO particles.33 Progenitor cells were cultured in RPMI 1640 medium (GIBCO, U.K.) with 10% FBS, 1% glutamine, and 100 U of penicillin/streptomycin at 37 °C in a 0.5% CO2 atmosphere. Cell labeling was initiated by removal of the cell culture medium from the adherent cells and by adding a new cell culture medium containing the modified USPIO particles. The cell-labeling efficiency was studied for different USPIO concentrations in the growth medium (0.03, 0.3, and 3 µmol of Fe/mL) at a fixed incubation time (6 h) or, alternatively, for different incubation times (20 min, 3 h, 6 h) at a constant USPIO concentration (0.03 µmol of Fe/mL) in the growth medium. After incubation the amount of iron left in the culture medium was determined, and the cells were washed twice with PBS to remove unbound particles. Following the washing step, the cells were trypsinized, removed from the culture dishes, and resuspended in a volume of 10 mL of culture medium. For each condition 500 µL of the cell suspension was recultivated for 24 h in 24-well plates with glass coverslips for subsequent Prussian blue staining. A 30 µL volume of the suspension was used for Trypan blue staining. The residual cell suspensions were used for ICP-MS and for MR relaxometry of the cell pellets. Cell Viability. The toxicity of the different USPIO particles on cells was analyzed using trypan blue (trypan blue stain, Incitrogen Corp., Grand Island, NY) and TUNEL (In Situ Cell Death Detection Kit, TMR red, Roche Diagnostics GmbH, Mannheim, Germany) staining and MTS analysis (CellTiter 96 AQeous nonradioactive cell proliferation assay, Promega Corp., Woods Hollow Rd., Madison, WI). For this purpose, the cells were incubated for 6 and 24 h with USPIO particles at concentrations of 0.03, 0.3, and 3.0 µmol/mL. Trypan blue positive cells were determined using a Neubauer counting chamber. TUNEL staining was performed for cells grown on glass coverslides according to the instructions of the manufacturers. Hereby five vision fields were analyzed at 40-fold magnification using an Olympus microscope (AX70). The percentage of positive cells as a function of the total cell number was determined. The MTS assay was performed using cells grown in a 96-well plate following the protocol given by the manufacturer, and the absorbance at 490 nm was recorded using an ELISA plate reader. Prussian Blue Staining. After adhesion of reincubated cells to the glass coverslips, the coverslips were washed three times with phosphate-buffered saline. Subsequently, the cells were fixed with a 1:1 mixture of methanol (-20 °C) and acetone. Staining was performed by adding 10% potassium ferrocyanide for 5 min and 10% potassium ferrocyanide in 20% hydrochloric acid for 30 min. Finally the glass coverslips were washed with PBS and counterstained with nuclear fast red. Determination of the Intracellular Iron Contents. The amount of iron present in the cells was determined by high-resolution sector field ICP-MS (Element 2, Thermo Electron, Bremen, Germany) equipped with an autosampler (ASX-100, CETAC, Omaha, NE). Data were acquired at medium resolution (4000) using rhodium (5 ppb) as an internal standard. A PFA spray chamber equipped with a PFA 100 microflow nebulizer (CETAC) was used for sample introduction. The instrument was tuned and calibrated via infusion of a 1 ng/mL multielement standard solution (Merck, Darmstadt, Germany). Digestion of the cells was performed in a closed vessel microwave reaction system (CEM, Kamp-Linfort, Germany) after addition of 53% nitric acid (60%) at 3 bar and 600 W for 50 min. Samples were diluted 1:200 in H2O. Calibration was linear between 2 and 150 ng/mL iron (r > 0.999). The iron content was expressed in picograms of iron per cell. MR Relaxometry of the Cell Pellets. MR measurements were performed with cell pellets in Eppendorf tubes after removal of the supernatant (3 × 106 cells/tube). With respect to the ultrashort T2 relaxation times of the cell pellets a T2w spin-echo sequence, which covered lower TE values (TR ) 600 ms, TE range 4-22 ms, 16 (33) Sun, R.; Dittrich, J.; Le-Huu, M.; Mueller, M. M.; Bedke, J.; Kartenbeck, J.; Lehman, J. K.; Krueger, R.; Bock, M.; Huss, R.; Seliger, C.; Gro¨ne, H. J.; Misselwitz, B.; Semmler, W.; Kiessling, F. InVest. Radiol. 2005, 40, 504-513.

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Figure 1. X-ray powder diffraction patterns of (a) uncoated, (b) silica-coated, (c) APTMS-coated, and (d) AEAPTMS-coated USPIO particles. echoes, FOV ) 134 × 67 mm, matrix 128 × 64, slice thickness 10 mm, BW ) 781 Hz, NEX ) 5), was used to determine the T2 relaxation times. The determination of the T1 relaxation times was performed as described for the USPIO suspensions. MR Relaxometry and MRI of Labeled Cells in Gelatin. In this experiment it was investigated how different concentrations of USPIO-labeled cells alter the T2 relaxation times of a gelatin gel. For this purpose, the cells were labeled with silica-, APTMS- and AEAPTMS-coated USPIO particles (iron concentration of 3 µmol/ mL for 6 h). Subsequently, the cells were dispersed in 300 µL of gelatin at different concentrations (1 × 104, 5 × 104, 10 × 104, and 50 × 104 cells/mL). The T2 relaxation times of the gels containing the labeled cells were measured using a Carr-Purcell-MeiboomGill pulse sequence (TR ) 1200 ms, TE range 7.4-236.8 ms, 32 echoes, FOV ) 180 mm, matrix 39 × 128, slice thickness 10 mm, voxel size 1.9 × 1.4 × 10.0 mm, flip angle 180°, average 4). Morphological imaging was performed using a T2*-weighted FLASH 2D sequence (TR ) 213 ms, TE ) 10.9 ms, FOV ) 130 mm, matrix 112 × 256, slice thickness 1.5 mm, voxel size 0.5 × 0.5 × 1.5 mm, flip angle 10°, average 15). Statistical Evaluation. Statistical analysis of ICP-MS data and T2 relaxometry of cells incubated with the three different USPIO particles and of gelatin gels containing different amounts of labeled cells were conducted using a Student’s t test. A p value of