Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents

PURPOSE: To label mammalian and stem cells by combining commercially available transfection agents (TAs) with superparamagnetic iron oxide (SPIO) magnetic resonance (MR) imaging contrast agents. MATERIALS AND METHODS: Three TAs were incubated with ferumoxides and MION-46L in cell culture medium at various concentrations. Human mesenchymal stem cells, mouse lymphocytes, rat oligodendrocyte progenitor CG-4 cells, and human cervical carcinoma cells were incubated 2-48 hours with 25 microg of iron per milliliter of combined TAs and SPIO. Cellular labeling was evaluated with T2 relaxometry, MR imaging of labeled cell suspensions, and Prussian blue staining for iron assessment. Proliferation and viability of mesenchymal stem cells and human cervical carcinoma cells labeled with a combination of TAs and ferumoxides were evaluated. RESULTS: When ferumoxides-TA or MION-46L-TA was used, intracytoplasmic particles stained with Prussian blue stain were detected for all cell lines with a labeling efficiency of nearly 100%. Limited or no uptake was observed for cells incubated with ferumoxides or MION-46L alone. For TA-SPIO-labeled cells, MR images and relaxometry findings showed a 50%-90% decrease in signal intensity and a more than 40-fold increase in T2s. Cell viability varied from 103.7% +/- 9 to 123.0% +/- 9 compared with control cell viability at 9 days, and cell proliferation was not affected by endosomal incorporation of SPIO nanoparticles. Iron concentrations varied with ferumoxides-TA combinations and cells with a maximum of 30.1 pg +/- 3.7 of iron per cell for labeled mesenchymal stem cells. CONCLUSION: Magnetic labeling of mammalian cells with use of ferumoxides and TAs is possible and may enable cellular MR imaging and tracking in experimental and clinical settings.     

Comparison of two superparamagnetic viral-sized iron oxide particles ferumoxides and ferumoxtran-10 with a gadolinium chelate in imaging intracranial tumors

BACKGROUND AND PURPOSE: Ultrasmall superparamagnetic iron oxide particles result in shortening of T1 and T2 relaxation time constants and can be used as MR contrast agents. We tested four hypotheses by evaluating MR images of intracranial tumors after infusion of two iron oxide agents in comparison with a gadolinium chelate: 1) Ferumoxtran in contrast to ferumoxides can be used as an intravenous MR contrast agent in intracranial tumors; 2) ferumoxtran enhancement, albeit delayed, is similar to gadolinium enhancement; 3) ferumoxtran-enhanced MR images in contrast to gadolinium-enhanced MR images may be compared with histologic specimens showing the cellular location of iron oxide particles; 4) ferumoxtran can serve as a model for viral vector delivery. METHODS: In 20 patients, ferumoxides and ferumoxtran were intravenously administered at recommended clinical doses. MR imaging was performed 30 minutes and 4 hours after ferumoxides infusion (n = 3), whereas ferumoxtran-enhanced MR imaging (n = 17) was performed 6 and 24 hours after infusion in the first five patients and 24 hours after infusion in the remaining 12. MR sequences were spin-echo (SE) T1-weighted, fast SE T2- and proton density-weighted, gradient-recalled-echo T2*-weighted, and, in four cases, echo-planar T2-weighted sequences. Representative regions of interest were chosen on pre- and postcontrast images to compare each sequence and signal intensity. RESULTS: Despite some degree of gadolinium enhancement in all tumors, no significant T1 or T2 signal intensity changes were seen after ferumoxides administration at either examination time. Fifteen of 17 patients given ferumoxtrans had T1 and/or T2 shortening consistent with iron penetration into tumor. Histologic examination revealed minimal iron staining of the tumor with strong staining at the periphery of the tumors. CONCLUSION: 1) Ferumoxtran can be used as an intravenous MR contrast agent in intracranial tumors, mostly malignant tumors. 2) Enhancement with ferumoxtran is comparable to but more variable than that with the gadolinium chelate. 3) Histologic examination showed a distribution of ferumoxtran particles similar to that on MR images, but at histology the cellular uptake was primarily by parenchymal cells at the tumor margin. 4) Ferumoxtran may be used as a model for viral vector delivery in malignant brain tumors.     

Antimyosin-labeled monocrystalline iron oxide allows detection of myocardial infarct: MR antibody imaging.

The synthesis and in vivo antigen targeting of a novel iron oxide compound were studied. A monocrystalline iron oxide nanoparticle (MION) was synthesized that contains a small (mean diameter, 2.9 nm +/- 0.9) single crystal core, passes through capillary membranes, and exhibits superparamagnetism. The MION was attached to antimyosin Fab (R11D10) and used for immunospecific magnetic resonance (MR) imaging of cardiac infarcts One hour after intravenous administration of MION-R11D10 in rats (100 mumol/kg), a marked decrease in the signal intensity of infarcted myocardium was observed. Immunohistochemical correlation confirmed the specific binding of the immunoconjugate to infarcted, but not to normal, myocardium. No decrease in cardiac signal intensity was observed when unconjugated MION was administered intravenously. The results indicate the feasibility of immunospecific MR imaging in living organisms.     

Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular MR imaging

PURPOSE: To evaluate the effect of using the ferumoxides-poly-l-lysine (PLL) complex for magnetic cell labeling on the long-term viability, function, metabolism, and iron utilization of mammalian cells. MATERIALS AND METHODS: PLL was incubated with ferumoxides for 60 minutes, incompletely coating the superparamagnetic iron oxide (SPIO) through electrostatic interactions. Cells were coincubated overnight with the ferumoxides-PLL complex, and iron uptake, cell viability, apoptosis indexes, and reactive oxygen species formation were evaluated. The disappearance or the life span of the detectable iron nanoparticles in cells was also evaluated. The iron concentrations in the media also were assessed at different time points. Data were expressed as the mean +/- 1 SD, and one-way analysis of variance and the unpaired Student t test were used to test for significant differences. RESULTS: Intracytoplasmic nanoparticles were stained with Prussian blue when the ferumoxides-PLL complex had magnetically labeled the human mesenchymal stem and HeLa cells. The long-term viability, growth rate, and apoptotic indexes of the labeled cells were unaffected by the endosomal incorporation of SPIO, as compared with these characteristics of the nonlabeled cells. In nondividing human mesenchymal stem cells, endosomal iron nanoparticles could be detected after 7 weeks; however, in rapidly dividing cells, intracellular iron had disappeared by five to eight divisions. A nonsignificant transient increase in reactive oxygen species production was seen in the human mesenchymal stem and HeLa cell lines. Labeled human mesenchymal stem cells did not differentiate to other lineage. A significant increase in iron concentration was observed in both the human mesenchymal stem and HeLa cell media at day 7. CONCLUSION: Magnetic cellular labeling with the ferumoxides-PLL complex had no short- or long-term toxic effects on tumor or stem cells.     

Early identification of aortic valve sclerosis using iron oxide enhanced MRI

Purpose:

To test the ability of MION‐47 enhanced MRI to identify tissue macrophage infiltration in a rabbit model of aortic valve sclerosis (AVS).

Materials and Methods:

The aortic valves of control and cholesterol‐fed New Zealand White rabbits were imaged in vivo pre‐ and 48 h post‐intravenous administration of MION‐47 using a 1.5 Tesla (T) MR clinical scanner and a CINE fSPGR sequence. MION‐47 aortic valve cusps were imaged ex vivo on a 3.0T whole‐body MR system with a custom gradient insert coil and a three‐dimensional (3D) FIESTA sequence and compared with aortic valve cusps from control and cholesterol‐fed contrast‐free rabbits. Histopathological analysis was performed to determine the site of iron oxide uptake.

Results:

MION‐47 enhanced the visibility of both control and cholesterol‐fed rabbit valves in in vivo images. Ex vivo image analysis confirmed the presence of significant signal voids in contrast‐administered aortic valves. Signal voids were not observed in contrast‐free valve cusps. In MION‐47 administered rabbits, histopathological analysis revealed iron staining not only in fibrosal macrophages of cholesterol‐fed valves but also in myofibroblasts from control and cholesterol‐fed valves.

Conclusion:

Although iron oxide labeling of macrophage infiltration in AVS has the potential to detect the disease process early, a macrophage‐specific iron compound rather than passive targeting may be required. J. Magn. Reson. Imaging 2010;31:110–116. © 2009 Wiley‐Liss, Inc.     

Polyclonal human immunoglobulin G labeled with polymeric iron oxide: antibody MR imaging

Human polyclonal immunoglobulin (Ig) G was attached to a monocrystalline iron oxide nanocompound (MION), a small superparamagnetic probe developed for receptor and antibody magnetic resonance (MR) imaging. The resulting complex, MION-IgG, had a slightly negative surface charge, a molecular weight of 150-180 kDa, and 0.36 microgram of IgG attached per milligram of iron. After intravenous administration of MION-IgG to normal rats, most of the compound localized in liver, spleen, and bone marrow. In an animal model of myositis, MION-IgG caused reduced signal intensity (most apparent on T2-weighted spin-echo and gradient-echo images) at the site of inflammation. No change in signal intensity existed after an injection of unlabeled MION. Site-specific localization of MION-IgG was corroborated with scintigraphic imaging with indium-111 IgG and MION-In-111-IgG and was confirmed histologically with iron staining. These results indicate that antibody MR imaging is feasible in vivo. Target-specific and antibody MR imaging could be easily extended to other applications, including detection of cancer, infarction, and degenerative diseases.     

Uptake of dextran-coated monocrystalline iron oxides in tumor cells and macrophages

Although several dextran-coated iron oxide preparations are in preclinical and clinical use, little is known about the mechanism of uptake into cells. As these particles have been shown to accumulate in macrophages and tumor cells, we performed cellular uptake and inhibition studies with a prototypical monocrystalline iron oxide nanoparticle (MION). MION particles were labeled with fluorescein isothiocyanate or radioiodinated and purified by gel permeation chromatography. Two preparations of MION particles were used in cell experiments: nontreated MION and plasma-opsonized MION purified by gradient density purification. As determined by immunoblotting, opsonization resulted in C3, vitro-nectin, and fibronectin association with MION. Incubation of cells with fluorescent MION showed active uptake of particles in macrophages both before and after opsonization. In C6 tumor cells, however, intracellular MION was only detectable in dividing cells. Quantitatively, ¹²⁵I-labeled MION was internalized into cells with uptake values ranging from 17 ng (in 9L gliosarcoma) to 970 ng iron per million cells for peritoneal macrophages. Opsonization increased MION uptake into macrophages sixfold, whereas it increased the uptake in C6 tumor cells only twofold. Results from uptake inhibition assay suggest that cellular uptake of nonopsonized (dextran-coated) MION particles is mediated by fluid-phase endocytosis, whereas receptor-mediated endocytosis is presumably responsible for the uptake of opsonized (protein-coated) particles.     

Gene expression profiling reveals early cellular responses to intracellular magnetic labeling with superparamagnetic iron oxide nanoparticles

With MRI (stem) cell tracking having entered the clinic, studies on the cellular genomic response toward labeling are warranted. Gene expression profiling was applied to C17.2 neural stem cells following superparamagnetic iron oxide/PLL (poly‐L ‐lysine) labeling over the course of 1 week. Relative to unlabeled cells, less than 1% of genes (49 total) exhibited greater than 2‐fold difference in expression in response to superparamagnetic iron oxide/PLL labeling. In particular, transferrin receptor 1 (Tfrc) and heme oxygenase 1 (Hmox1) expression was downregulated early, whereas genes involved in lysosomal function (Sulf1) and detoxification (Clu, Cp, Gstm2, Mgst1) were upregulated at later time points. Relative to cells treated with PLL only, cells labeled with superparamagnetic iron oxide/PLL complexes exhibited differential expression of 1399 genes. Though these differentially expressed genes exhibited altered expression over time, the overall extent was limited. Gene ontology analysis of differentially expressed genes showed that genes encoding zinc‐binding proteins are enriched after superparamagnetic iron oxide/PLL labeling relative to PLL only treatment, whereas members of the apoptosis/programmed cell death pathway did not display increased expression. Overexpression of the differentially expressed genes Rnf138 and Abcc4 were confirmed by quantitative real‐time polymerase chain reaction. These results demonstrate that, although early reactions responsible for iron homeostasis are induced, overall neural stem cell gene expression remains largely unaltered following superparamagnetic iron oxide/PLL labeling. Magn Reson Med 63:1031–1043, 2010. © 2010 Wiley‐Liss, Inc.     

MRI detection of macrophages labeled using micrometer-sized iron oxide particles

PURPOSE: To evaluate cellular labeling of immune cells using micron-sized iron oxide particles (MPIOs) and evaluate the MR relaxivity and MRI detection of the labeled cells. MATERIALS AND METHODS: Immune cells isolated from mice and rats were labeled with three different sizes of MPIO particles (0.35, 0.90, or 1.63 microm). These labeled cells were characterized using transmission electron microscopy (TEM), fluorescence microscopy, flow cytometry, MR relaxometry, and MRI. RESULTS: Macrophage uptake of MPIOs was found to be highest for the 1.63-microm size particles. MR relaxivity measurements indicated greater spin-spin relaxation for MPIO-labeled cells relative to cells labeled with nanometer-sized ultra-small superparamagnetic iron oxide (USPIO) particles with similar iron content. TEM and fluorescence microscopy indicated cellular uptake of multiple MPIO particles per cell. Macrophages labeled with 1.63-microm MPIOs had an average cellular iron uptake of 39.1 pg/cell, corresponding to approximately 35 particles per cell. CONCLUSION: Cells labeled with one or more MPIO particles could be readily detected ex vivo at 11.7 Tesla and after infusion of the MPIO-labeled macrophages into the kidney of a rat, hypointense regions of the outer cortex are observed, in vivo, by MRI at 4.7 Tesla.     

Magnetically labeled cells can be detected by MR imaging

To determine the feasibility of MR imaging of magnetically labeled cells, different cell lines were labeled with monocrystalline iron oxide (MION) particles. Phantoms containing MION labeled cells were then assembled and imaged by MR at 1.5 T using T1-weighted and T2-weighted pulse sequences. MION uptake ranged from 8.5 × 10⁴ to 2.9 × 10⁵ particles/cell for tumor cells (9L and LX1, respectively) to 1.5 × 10⁶ to 4.8 × 10⁸ particles/cell for “professional phagocytes” (J774 and peritoneal macrophages, respectively). On the T1-weighted images, cell-internalized MION appeared hyperintense relative to agar and similar to MION in aqueous solution. On T2-weighted images, signal intensity varied according to concentration of MION within cells. Cell-internalized MION caused similar MR signal changes of cells as did free MION; however, at a dose that was an order of magnitude lower, depending on the pulse sequence used. The detectability of MION within cells was approximately 2 ng Fe, which corresponded to 10⁵ tumor cells/well or 5 × 10³ macrophages/well. We conclude that a variety of cells can be efficiently labeled with MION by simple incubation. Intracellular labeling may be used for MR imaging of in vivo cell tracking.     

Enhancement of gas‐filled microbubble R2* by iron oxide nanoparticles for MRI

Gas‐filled microbubbles have the potential to become a unique intravascular MR contrast agent due to their magnetic susceptibility effect, biocompatibility, and localized manipulation via ultrasound cavitation. However, microbubble susceptibility effect is relatively weak when compared with other intravascular MR susceptibility contrast agents. In this study, enhancement of microbubble susceptibility effect by entrapping monocrystalline iron oxide nanoparticles (MIONs) into polymeric microbubbles was investigated at 7 T in vitro. Apparent T₂ enhancement (ΔR₂*) induced by microbubbles was measured to be 79.2 ± 17.5 sec^?1 and 301.2 ± 16.8 sec^?1 for MION‐free and MION‐entrapped polymeric microbubbles at 5% volume fraction, respectively. ΔR₂* and apparent transverse relaxivities (r₂*) for MION‐entrapped polymeric microbubbles and MION‐entrapped solid microspheres (without gas core) were also compared, showing the synergistic effect of the gas core with MIONs. This is the first experimental demonstration of microbubble susceptibility enhancement for MRI application. This study indicates that gas‐filled polymeric microbubble susceptibility effect can be substantially increased by incorporating iron oxide nanoparticles into microbubble shells. With such an approach, microbubbles can potentially be visualized with higher sensitivity and lower concentrations by MRI. Magn Reson Med, 2010. © 2009 Wiley‐Liss, Inc.     

Comparison of iron oxide labeling properties of hematopoietic progenitor cells from umbilical cord blood and from peripheral blood for subsequent in vivo tracking in a xenotransplant mouse model XXX

RATIONALE AND OBJECTIVES: To compare and optimize ferumoxides labeling of human hematopoietic progenitor cells from umbilical cord blood and from peripheral blood for subsequent in vivo tracking with a clinical 1.5 T MR scanner. MATERIALS AND METHODS: Human hematopoietic progenitor cells, derived from umbilical cord blood or peripheral blood, were labeled with Ferumoxides by simple incubation or lipofection. Cellular iron uptake was quantified with spectrometry. Then, 3 x 10(7)-labeled cells were injected into the tail vein of 12 female nude Balb/c mice. The mice underwent magnetic resonance imaging before and 24 hours after injection. Precontrast and postcontrast signal intensities of liver, spleen, and bone marrow were measured and tested for significant differences with the t-test. Immunostains served as a histopathologic standard of reference. RESULTS: After labeling by simple incubation, only umbilical cord blood cells, but not peripheral blood cells, showed a significant iron uptake and could be tracked in vivo with magnetic resonance imaging. Using lipofection, both cell types could be tracked in vivo. A significant decline in signal intensity was observed in liver, spleen, and bone marrow at 24 hours after injection of efficiently labeled ferumoxides cells (P < .05). Histopathology proved the distribution of iron oxide-labeled cells to these organs. CONCLUSION: Hematopoietic progenitor cells from umbilical cord blood can be labeled by simple incubation with an Food and Drug Administration-approved magnetic resonance contrast agent with sufficient efficiency to provide an in vivo cell tracking at 1.5 T. Progenitor cells from peripheral blood need to be labeled with adjunctive transfection techniques to be depicted in vivo at 1.5 T.     

Dynamic liver imaging with iron oxide agents: Effects of size and biodistribution on contrast

In vivo effective relaxation rates in normal rat liver were evaluated for four dextran coated iron oxide agents: monocrystal-line iron oxide nanocolloid (MION) with a mean particle diameter of 3.9 nm, a polycrystalline agent (PION) with a larger mean diameter of 12 nm, and these two agents labeled with the asialofetuin (ASF) protein for high hepatocytic receptor binding affinity (MION-ASF and PION-ASF). Using echo planar imaging at 2 Tesla, dose response was measured with high temporal resolution for 3 h after injection of agent, and by comparing with relaxivities in vitro and in brain, dominant in vivo contrast phenomena were elucidated. While transverse relaxivity for PION-ASF exceeded that for MION-ASF by almost a factor of 2 in solution, relaxation rates in vivo became equivalent. Liver relaxation using non-ASF agents was consistent with rapid water exchange between vascular and extravascular compartments, which dominated relaxation as a result of agent accumulation in Kupffer cells.     

A physiological barrier distal to the anatomic blood-brain barrier in a model of transvascular delivery

BACKGROUND AND PURPOSE: Osmotic disruption of the blood-brain barrier (BBB) provides a method for transvascular delivery of therapeutic agents to the brain. The apparent global delivery of viral-sized iron oxide particles to the rat brain after BBB opening as seen on MR images was compared with the cellular and subcellular location and distribution of the particles. METHODS: Two dextran-coated superparamagnetic monocrystalline iron oxide nanoparticle contrast agents, MION and Feridex, were administered intraarterially in rats at 10 mg Fe/kg immediately after osmotic opening of the BBB with hyperosmolar mannitol. After 2 to 24 hours, iron distribution in the brain was evaluated first with MR imaging then by histochemical analysis and electron microscopy to assess perivascular and intracellular distribution. RESULTS: After BBB opening, MR images showed enhancement throughout the disrupted hemisphere for both Feridex and MION. Feridex histochemical staining was found in capillaries of the disrupted hemisphere. Electron microscopy showed that the Feridex particles passed the capillary endothelial cells but did not cross beyond the basement membrane. In contrast, after MION delivery, iron histochemistry was detected within cell bodies in the disrupted hemisphere, and the electron-dense MION core was detected intracellularly and extracellularly in the neuropil. CONCLUSION: MR images showing homogeneous delivery to the brain at the macroscopic level did not indicate delivery at the microscopic level. These data support the presence of a physiological barrier at the basal lamina, analogous to the podocyte in the kidney, distal to the anatomic (tight junction) BBB, which may limit the distribution of some proteins and viral particles after transvascular delivery to the brain.     

Superparamagnetic iron oxide: pharmacokinetics and toxicity

The pharmacokinetics (distribution, metabolism, bioavailability, excretion) and toxicity (acute and subacute toxicity, mutagenicity) of a superparamagnetic iron oxide preparation (AMI-25), currently used in clinical trials, were evaluated by 59Fe radiotracer studies, measurements of relaxation times, the ability to reverse iron deficiency anemia, histologic examination, and laboratory parameters. One hour after administration of AMI-25 to rats (18 mumol Fe/kg; 1 mg Fe/kg), 82.6 +/- 0.3% of the administered dose was sequestered in the liver and 6.2 +/- 7.6% in the spleen. Peak concentrations of 59Fe were found in liver after 2 hr and in the spleen after 4 hr. 59Fe slowly cleared from liver (half-life, 3 days) and spleen (half-life, 4 days) and was incorporated into hemoglobin of erythrocytes in a time-dependent fashion. The half-time of the T2 effect on liver and spleen (24-48 hr) was shorter than the 59Fe clearance, indicating metabolism of AMI-25 into other forms of iron. IV administration of AMI-25 (30 mg Fe/kg) corrected iron-deficiency anemia and showed bioavailability similar to that of commercially available IV iron preparations within 7 days. No acute or subacute toxic effects were detected by histologic or serologic studies in rats or beagle dogs who received a total of 3000 mumol Fe/kg, 150 times the dose proposed for MR imaging of the liver. Our results indicate that AMI-25 is a fully biocompatible potential contrast agent for MR.     

Superparamagnetic iron oxide contrast agents: physicochemical characteristics and applications in MR imaging

Superparamagnetic iron oxide MR imaging contrast agents have been the subjects of extensive research over the past decade. The iron oxide particle size of these contrast agents varies widely, and influences their physicochemical and pharmacokinetic properties, and thus clinical application. Superparamagnetic agents enhance both T1 and T2/T2* relaxation. In most situations it is their significant capacity to reduce the T2/T2* relaxation time to be utilized. The T1 relaxivity can be improved (and the T2/T2* effect can be reduced) using small particles and T1-weighted imaging sequences. Large iron oxide particles are used for bowel contrast [AMI-121 (i.e. Lumirem and Gastromark) and OMP (i.e. Abdoscan), mean diameter no less than 300 nm] and liver/spleen imaging [AMI-25 (i.e. Endorem and Feridex IV, diameter 80-150 nm); SHU 555A (i.e. Resovist, mean diameter 60 nm)]. Smaller iron oxide particles are selected for lymph node imaging [AMI-227 (i.e. Sinerem and Combidex, diameter 20-40 nm)], bone marrow imaging (AMI-227), perfusion imaging [NC100150 (i.e. Clariscan, mean diameter 20 nm)] and MR angiography (NC100150). Even smaller monocrystalline iron oxide nanoparticles are under research for receptor-directed MR imaging and magnetically labeled cell probe MR imaging. Iron oxide particles for bowel contrast are coated with insoluble material, and all iron oxide particles for intravenous injection are biodegradable. Superparamagnetic agents open up an important field for research in MR imaging.     

Targeting of hematopoietic progenitor cells with MR contrast agents

PURPOSE: To label human hematopoietic progenitor cells with various magnetic resonance (MR) imaging contrast agents and to obtain 1.5-T MR images of them. MATERIALS AND METHODS: Hematopoietic progenitor cells, labeled with ferumoxides, ferumoxtran, magnetic polysaccharide nanoparticles-transferrin, P7228 liposomes, and gadopentetate dimeglumine liposomes underwent MR imaging with T1- and T2-weighted spin-echo and fast field-echo sequences. Data were analyzed by measuring MR signal intensities and R1 and R2* relaxation rates of labeled cells and nonlabeled control cells. Mean quantitative data for the various contrast agent groups were assessed for significant differences compared with control cells by means of the Scheffe test. As a standard of reference, MR imaging data were compared with electron microscopic and spectrometric data. RESULTS: For all contrast agents, intracellular cytoplasm uptake was demonstrated with electron microscopy and was quantified with spectrometry. When compared with nonlabeled control cells, progenitor cells labeled with iron oxides showed significantly (P <.05) increased R2*. Cells labeled with gadopentetate dimeglumine liposomes showed significantly increased R1. Detection thresholds were 5 x 10(5) cells for gadopentetate dimeglumine liposomes and ferumoxtran, 2.5 x 10(5) cells for ferumoxides and P7228 liposomes, and 1 x 10(5) cells for magnetic polysaccharide nanoparticles-transferrin. CONCLUSION: Hematopoietic progenitor cells can be labeled with MR contrast agents and can be depicted with a standard 1.5-T MR imager.     

Use of magnetic nanoparticles to monitor alginate‐encapsulated βTC‐tet cells

Noninvasive monitoring of tissue‐engineered constructs is an important component in optimizing construct design and assessing therapeutic efficacy. In recent years, cellular and molecular imaging initiatives have spurred the use of iron oxide‐based contrast agents in the field of NMR imaging. Although their use in medical research has been widespread, their application in tissue engineering has been limited. In this study, the utility of monocrystalline iron oxide nanoparticles (MIONs) as an NMR contrast agent was evaluated for βTC‐tet cells encapsulated within alginate/poly‐L‐lysine/alginate (APA) microbeads. The constructs were labeled with MIONs in two different ways: 1) MION‐labeled βTC‐tet cells were encapsulated in APA beads (i.e., intracellular compartment), and 2) MION particles were suspended in the alginate solution prior to encapsulation so that the alginate matrix was labeled with MIONs instead of the cells (i.e., extracellular compartment). The data show that although the location of cells can be identified within APA beads, cell growth or rearrangement within these constructs cannot be effectively monitored, regardless of the location of MION compartmentalization. The advantages and disadvantages of these techniques and their potential use in tissue engineering are discussed. Magn Reson Med 61:282–290, 2009. © 2009 Wiley‐Liss, Inc.     

MR imaging of in vivo recruitment of iron oxide-labeled macrophages in experimentally induced soft-tissue infection in mice

PURPOSE: To evaluate the feasibility of magnetic resonance (MR) imaging in depicting in vivo recruitment of iron oxide-labeled macrophages in experimentally induced soft-tissue infection. MATERIALS AND METHODS: The study was performed according to the guidelines of the U.S. National Institutes of Health and recommendations of the committee on animal research. The protocol was approved by the local institutional review committee on animal care. Experimental soft-tissue infection in 12 mice was induced by inoculation with a 5 x 10(7) colony-forming units of Staphylococcus aureus into the left calf. Peritoneal macrophages were harvested from thioglycollate-treated mice, cultured, and labeled with iron oxide in vitro. The iron oxide-labeled macrophage (macrophage group, n = 6) or iron oxide solution (control group, n = 6) was administered through the tail vein. The left calf of the mice was imaged on days 2 and 3 with a 4.7-T MR unit. Changes in relative signal intensity (SI) and pattern of contrast material enhancement (macrophage distribution) were analyzed and compared with histopathologic findings. Statistical analysis was performed with the Wilcoxon matched-pairs signed rank test. RESULTS: On MR images obtained 24 hours after administration of macrophage labeled with iron oxide, a band-shaped lower SI zone was noted in the abscess wall, which corresponded to the distribution of the iron oxide-labeled macrophages at histopathologic examination. The relative SI of the abscess wall significantly decreased after injection of iron oxide-labeled macrophages (median, 0.42) compared with that before injection (median, 1.23) (P = .031). In the control group, the SI change after administration of iron oxide solution was not significant (P = .688). CONCLUSION: Homing of intravenously administered iron oxide-labeled macrophages can be monitored with MR imaging and may provide a tool to investigate interactions between macrophages and the invading pathogens.     

Transferrin receptor upregulation: in vitro labeling of rat mesenchymal stem cells with superparamagnetic iron oxide

PURPOSE: To prospectively evaluate the influence of superparamagnetic iron oxide (SPIO) or ultrasmall SPIO (USPIO) particles on the surface epitope pattern of adult mesenchymal stem cells (MSCs) by regulating the expression of transferrin receptor and to prospectively evaluate the influence of transfection agents (TAs) on the uptake of SPIO or USPIO particles in MSCs. MATERIALS AND METHODS: The study was approved by the institutional animal care committee of the University of Tübingen. MSCs were isolated from the bone marrow of four rats. To obtain highly homogeneous MSC populations, MSCs from one rat were single-cell cloned. One MSC clone was characterized and selected for the labeling experiments. The MSCs, which were characterized with flow cytometry and in vitro differentiation, were labeled with 200 microg/mL SPIO or USPIO or with 60 microg/mL SPIO or USPIO in combination with TAs. Aggregations of labeled cells were accommodated inside a defined volume in an agar gel matrix. Magnetic resonance (MR) imaging was performed to measure SPIO- or USPIO-induced signal voids. Quantification of cellular total iron load (TIL) (intracellular iron plus iron coating the cellular surface), determination of cellular viability, and electron microscopy were also performed. RESULTS: Labeling of MSCs with SPIO or USPIO was feasible without affecting cell viability (91.1%-94.7%) or differentiation potential. For MR imaging, SPIO plus a TA was most effective, depicting 5000 cells with an average TIL of 76.5 pg per cell. SPIO or USPIO particles in combination with TAs coated the cellular surface but were not incorporated into cells. In nontransfected cells, SPIO or USPIO was taken up. MSCs labeled with SPIO or USPIO but without a TA showed enhanced expression of transferrin receptor, in contrary to both MSCs labeled with SPIO or USPIO and a TA and control cells. CONCLUSION: SPIO or USPIO labeling without TAs has an influence on gene expression of MSCs upregulating transferrin receptor. Furthermore, SPIO labeling with a TA will coat the cellular surface.