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.     

Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro

To evaluate the capacity of human monocytes to phagocytose various approved iron oxide based magnetic resonance (MR) contrast agents and to optimize in vitro labeling of these cells. Human monocytes were incubated with two superparamagnetic iron oxide particles (SPIO) as well as two ultrasmall SPIO (USPIO) at varying iron oxide concentrations and incubation times. Iron uptake in monocytes was proven by histology, quantified by atomic emission absorption spectrometry and depicted with T2* weighted fast field echo (FFE) MR images at 1.5 T. Additionally, induction of apoptosis in iron oxide labeled monocytes was determined by YO-PRO-1 staining. Cellular iron uptake was significantly (P<0.01) higher after incubation with SPIO compared with USPIO. For SPIO, the iron oxide uptake was significantly (P<0.01) higher after incubation with the ionic Ferucarbotran as compared with the non-ionic Ferumoxides. Efficient cell labeling was achieved after incubation with Ferucarbotran at concentrations 500 g Fe/ml and incubation times 1 h, resulting in a maximal iron oxide uptake of up to 50 pg Fe/cell without impairment of cell viability. In vitro labeling of human monocytes for MR imaging is most effectively obtained with the approved SPIO Ferucarbotran. Potential subsequent in vivo cell tracking applications comprise, e.g. specific targeting of inflammatory processes.     

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.     

Quantification of superparamagnetic iron oxide (SPIO)-labeled cells using MRI

PURPOSE: To show the feasibility of using magnetic resonance imaging (MRI) to quantify superparamagnetic iron oxide (SPIO)-labeled cells. MATERIALS AND METHODS: Lymphocytes and 9L rat gliosarcoma cells were labeled with ferumoxides-protamine sulfate complex (FE-PRO). The cells were labeled efficiently (more than 95%) and the iron concentration inside each cell was measured by spectrophotometry (4.77-30.21 pg). Phantom tubes containing different numbers of labeled or unlabeled cells, as well as different concentrations of FE-PRO, were made. In addition, labeled and unlabeled cells were injected into fresh and fixed rat brains. RESULTS: Cellular viability and proliferation of labeled and unlabeled cells were shown to be similar. T2-weighted images were acquired using 7T and 3T MRI systems, and R2 maps of the tubes containing cells, free FE-PRO, and brains were made. There was a strong linear correlation between R2 values and labeled cell numbers, but the regression lines were different for the lymphocytes and gliosarcoma cells. Similarly, there was strong correlation between R2 values and free iron. However, free iron had higher R2 values than the labeled cells for the same concentration of iron. CONCLUSION: Our data indicate that in vivo quantification of labeled cells can be done by careful consideration of different factors and specific control groups.     

Macrophage endocytosis of superparamagnetic iron oxide nanoparticles: mechanisms and comparison of ferumoxides and ferumoxtran-10

RATIONALE AND OBJECTIVES: Superparamagnetic iron oxides (SPIO) used as magnetic resonance (MR) contrast agents undergo specific uptake by macrophages. The purpose of this study was first to determine the mechanism of macrophage uptake for Ferumoxides by using competition experiments with specific ligands of scavenger receptors SR-A (I/II) and second, to evaluate and compare the internalization of 2 different contrast agents, Ferumoxides (SPIO) and Ferumoxtran-10 (USPIO: ultrasmall superparamagnetic iron oxide) using macrophages obtained by chemical activation of human monocytic cells. METHODS: Ferumoxides and Ferumoxtran-10 are 2 MR contrast agents, composed of dextran-coated iron oxide nanoparticles. The endocytosis pathway of Ferumoxides was studied using competition experiments on mouse peritoneal macrophages in the presence of specific ligands of scavenger receptors SR-A (types I and II): polyinosinic acid and fucoidan. In vitro assays using THP-1 (human promonocyte) cells activated into macrophages were performed in the presence of the 2 superparamagnetic nanoparticles. The cellular uptake was determined by measuring the iron content using ICP-AES (inductively coupled plasma-atomic emission spectrometry) and by Prussian blue staining. RESULTS: In the presence of polyinosinic acid or fucoidan, the endocytosis of Ferumoxides by mouse peritoneal macrophages was inhibited. This inhibition was obtained using 10 microg/mL of scavenger receptor ligands at a concentration of 62.5 microg Fe/mL of SPIO, and a dose-dependent relationship was observed. Without competitors, the percentage of uptake of Ferumoxides by mouse peritoneal macrophages ranged between 3 and 8%. On the human activated monocyte THP-1 cell assay, Ferumoxides underwent a higher macrophage uptake (between 1.1 and 3%) compared with Ferumoxtran-10 (between 0.03 and 0.12%). This difference is attributed to the larger size of Ferumoxides nanoparticles. CONCLUSIONS: Competition experiments indicate that the cellular uptake of Ferumoxides involves scavenger receptor SR-A-mediated endocytosis. The comparison between Ferumoxides and Ferumoxtran-10 confirms that macrophage uptake of iron oxide nanoparticles depends mainly on the size of these contrast agents.     

R2 and R2* mapping for sensing cell-bound superparamagnetic nanoparticles: in vitro and murine in vivo testing

PURPOSE: To prospectively determine the cellular iron uptake by using R2 and R2* mapping with multiecho readout gradient-echo and spin-echo sequences. MATERIALS AND METHODS: All experiments were approved by the institutional animal care committee. Lung carcinoma cells were lipofected with superparamagnetic iron oxides (SPIOs). Agarose gel phantoms containing (a) 1 x 10(5) CCL-185 cells per milliliter of agarose gel with increasing SPIO load (0.01-5.00 mg of iron per milliliter in the medium), (b) different amounts (5.0 x 10(3) to 2.5 x 10(5) cells per milliliter of agarose gel) of identically loaded cells, and (c) free (non-cell-bound) SPIOs at the iron concentrations described for (b) were analyzed with 3.0-T R2 and R2* relaxometry. Iron uptake was analyzed with light microscopy, quantified with atomic emission spectroscopy (AES), and compared with MR data. For in vivo relaxometry, agarose gel pellets containing SPIO-labeled cells, free SPIO, unlabeled control cells, and pure agarose gel were injected into three nude mice each. Linear and nonlinear regression analyses were performed. RESULTS: Light microscopy and AES revealed efficient SPIO particle uptake (mean uptake: 0.22 pg of iron per cell +/- 0.1 [standard deviation] for unlabeled cells, 31.17 pg of iron per cell +/- 4.63 for cells incubated with 0.5 mg/mL iron). R2 and R2* values were linearly correlated with cellular iron load, number of iron-loaded cells, and content of freely dissolved iron (r(2) range, 0.92-0.99; P < .001). For cell-bound SPIO, R2* effects were significantly greater than R2 effects (P < .01); for free SPIO, R2 and R2* effects were similar. In vivo relaxometry enabled accurate prediction of the number of labeled cells. R2' (R2* - R2) mapping enabled differentiation between cell-bound and free iron in vitro and in vivo. CONCLUSION: Quantitative R2 and R2* mapping enables noninvasive estimations of cellular iron load and number of iron-labeled cells. Cell-bound SPIOs can be differentiated from free SPIOs with R2' imaging.     

新型超顺磁性氧化铁标记SD大鼠脂肪干细胞的有效性及安全性研究

目的：采用新型超顺磁性氧化铁对 SD 大鼠来源的脂肪干细胞（ADSCs）进行标记,并与既往商用 SPIO 标记效果进行对比,探讨这种新型超顺磁性氧化铁标记的有效性及安全性。方法：分离、纯化、鉴定 SD 大鼠来源的 ADSCs,然后分不同浓度组（0、6、12、25、50和100μg／mL）和时间组（6、12、24和48 h）进行标记,通过普鲁士蓝染色测定铁标记率;在不影响细胞形态的前提下,对达到95％以上铁染色率的孵育浓度、时间进行标记安全性检测,包括活力、增殖力、细胞表面抗原表达;采用透射电子显微镜观察标记细胞的超微结构,采用 ICP-AES 对标记细胞内的铁含量进行测定,并与商用SPIO 标记效果进行对比。结果：在无细胞毒性的前提下,新型 SPIO 达到95％以上铁染色率的孵育浓度是12和25μg／mL,孵育时间是12 h;ICP-AES 检测显示具有表面正电荷的聚乙二醇（PEG）／聚乙烯亚胺（PEI）修饰的 SPIO 标记后细胞内的铁含量达到35．4 pg／cell（25μg／mL 中孵育12h 后）和20．16 pg／cell（12μg／mL 中孵育12h 后）,并随着孵育浓度的增加,细胞内的铁含量增加;而具有表面零电荷的 PEG／聚乙烯吡咯烷酮（PVP）修饰的 SPIO 标记后的铁含量仅为6．96 pg／cell（25μg／mL 中孵育12h 后）;透射电子显微镜显示标记后细胞器结构完整,内吸收的 SPIO 主要位于细胞质内的囊泡和溶酶体中。结论：新型 SPIO 在适当孵育浓度和时间下可以安全、快速标记 ADSCs;PEG／PEI 修饰的 SPIO 标记效果要远远比既往商用的 SPIO 快速有效,可作为一种优势的新型磁性标记物用于干细胞标记;而 PEG／PVP 修饰的SPIO 比起既往商用的 SPIO 并无明显优势,说明表面电荷在细胞标记中占有极其重要的角色。     

MR tracking of transplanted cells with "positive contrast" using manganese oxide nanoparticles

Rat glioma cells were labeled using electroporation with either manganese oxide (MnO) or superparamagnetic iron oxide (SPIO) nanoparticles. The viability and proliferation of SPIO-labeled cells (1.9 mg Fe/ml) or cells electroporated with a low dose of MnO (100 microg Mn/ml) was not significantly different from unlabeled cells; a higher MnO dose (785 microg Mn/ml) was found to be toxic. The cellular ion content was 0.1-0.3 pg Mn/cell and 4.4 pg Fe/cell, respectively, with cellular relaxivities of 2.5-4.8 s(-1) (R(1)) and 45-84 s(-1) (R(2)) for MnO-labeled cells. Labeled cells (SPIO and low-dose MnO) were each transplanted in contralateral brain hemispheres of rats and imaged in vivo at 9.4T. While SPIO-labeled cells produced a strong "negative contrast" due to the increase in R(2), MnO-labeled cells produced "positive contrast" with an increased R(1). Simultaneous imaging of both transplants with opposite contrast offers a method for MR "double labeling" of different cell populations.     

MRI in focal liver disease: A comparison of small and ultra-small superparamagnetic iron oxide as hepatic contrast agents

The purpose of this study was to compare small and ultrasmall superparamagnetic iron oxide particles (SPIO and USPIO, respectively) as MR contrast agents for the evaluation of focal hepatic disease. In two different patient groups (SPIO [n = 53], USPIO [n = 27]), with focal liver disease (metastases, hepatocellular carcinoma [HCC], hepatocellular adenoma [HCA], and focal nodular hyperplasia [FNH]), spin-echo T1- and T2-weighted images (T1WI, T2WI) were obtained at 1.0T, before and after intravenous contrast administration. The percentage signal-to-noise ratio (SNR) change and lesion-to-liver contrast (LLC) were measured and statistically compared. The liver decreased in signal intensity (SI) after SPIO administration (?28%) and increased after USPIO administration (+16%) on T1WI. On T2WI, the liver decreased in SI on postcontrast images with both agents (?78% SPIO, ?73% USPIO). This difference was not statistically significantly different (P ? .07). Both SPIO and USPIO provided >500% improvement in LLC on T2WI. On T1WI, LLC was increased in metastases (120%) and HCC (325%) with SPIO. Post-USPIO, LLC was increased on T1WI only in metastases (>500%). Both SPIO and USPIO show excellent hepatic uptake, presumed secondary to reticuloendothelial activity, based on the degree of %SI change seen in the liver after administration of contrast on T2WI. However, USPIO preparations exhibit blood pool activity that may aid in further characterization of focal liver lesions, as is evidenced by their greater T1 effect in the liver and in some focal liver lesions.     

Ultrasmall superparamagnetic iron oxide: an intravenous contrast agent for assessing lymph nodes with MR imaging

An ultrasmall superparamagnetic iron oxide (USPIO) preparation was evaluated as a potential intravenous contrast agent for lymph nodes. Relaxation time measurements and magnetic resonance (MR) imaging were performed in rats with normal lymph nodes and in rats with lymph node metastases. In normal animals, lymph node relaxation times decreased maximally within 24-48 hours after intravenous administration of USPIO. Twenty-four hours after administration, the T2 of normal lymph nodes had decreased from 74 msec +/- 2.2 to 30 msec +/- 0.7 (USPIO, 40 mumol of iron per kilogram) or 15 msec +/- 0.0 (200 mumol Fe/kg), whereas the T2 of metastatic nodes did not change. MR imaging of the animal model of nodal metastases confirmed the hypothesis that intravenously administered USPIO decreases signal intensity of normal but not metastatic nodes. A single intravenous administration of USPIO may allow detection of nodal metastases on the basis of signal intensity characteristics rather than the currently used, insensitive size characteristics.     

Application of the static dephasing regime theory to superparamagnetic iron-oxide loaded cells.

The relaxation rates of iron-oxide nanoparticles compartmentalized within cells were studied and found to satisfy predictions of the static dephasing (SD) regime theory. THP-1 cells in cell culture were loaded using two different iron-oxide nanoparticles (superparamagnetic iron-oxide (SPIO) and ultrasmall SPIO (USPIO)) with four different iron concentrations (0.05, 0.1, 0.2, and 0.3 mg/ml) and for five different incubation times (6, 12, 24, 36, and 48 hr). Cellular iron-oxide uptake was assessed using a newly developed imaging version of MR susceptometry, and was found to be linear with both dose and incubation time. R(2)* sensitivity to iron-oxide loaded cells was found to be 70 times greater than for R(2), and 3100 times greater than for R(1). This differs greatly from uniformly distributed nanoparticles and is consistent with a cellular bulk magnetic susceptibility (BMS) relaxation mechanism. The cellular magnetic moment was large enough that R(2)' relaxivity agreed closely with SD regime theory predictions for all cell samples tested [R(2)'=2 pi/(9 x the square root of 3) x gamma LMD] where the local magnetic dose (LMD) is the sample magnetization due to the presence of iron-oxide particles). Uniform suspensions of SPIO and USPIO produced R(2)' relaxivities that were a factor of 3 and 8 less, respectively, than SD regime theory predictions. These results are consistent with theoretical estimates of the required mass of iron per compartment needed to guarantee SD-regime-dominant relaxivity. For cellular samples, R(2) was shown to be dependent on both the concentration and distribution of iron-oxide particles, while R(2)' was sensitive to iron-oxide concentration alone. This work is an important first step in quantifying cellular iron content and ultimately mapping the density of a targeted cell population.     

Low‐intensity pulsed ultrasound increases cellular uptake of superparamagnetic iron oxide nanomaterial: Results from human osteosarcoma cell line U2OS

Purpose:

To determine whether low‐intensity pulsed ultrasound (LIPUS) is able to facilitate the uptake of a superparamagnetic iron oxide (SPIO) nanomaterial by cells that do not express high endocytosis capacity.

Materials and Methods:

The human osteosarcoma cell line U2OS and a silica‐coated SPIO functionalized peripherally with amines groups (overall diameter 8 nm) were used in this study. Adherent U2OS cells were labeled with SPIO by incubating with culture media containing the SPIO at 4.5 μg[Fe]/mL. LIPUS with the same parameters as those used in clinical application to accelerate bone fracture healing (1.5 MHz, duty cycle 1:4, spatial‐average temporal‐average intensity 30 mW/cm²) was applied to the cells at the beginning of the labeling process for 0, 0.5, 1, or 3 hours. The total incubation time with SPIO was 12 hours. SPIO labeling efficiency was evaluated with Prussian blue staining and a blueness measurement method, and magnetic resonance imaging (MRI) of cell pellets via measuring areas of SPIO‐induced signal void.

Results:

Both Prussian blue staining and in vitro MRI demonstrated that LIPUS application increased the SPIO nanomaterial labeling efficiency for U2OS cells in an exposure‐duration‐dependent manner.

Conclusion:

This study is a “proof of concept” that LIPUS can facilitate the cellular take‐up of SPIO nanomaterial. J. Magn. Reson. Imaging 2010;31:1508–1513. © 2010 Wiley‐Liss, Inc.     

Atherosclerotic imaging using 4 types of superparamagnetic iron oxides: New possibilities for mannan-coated particles

Purpose

We used magnetic resonance imaging (MRI) and histologic techniques to compare the uptake by the rabbit atherosclerotic wall of 4 types of superparamagnetic iron oxide (SPIO) particles, i.e. SPIO, mannan-coated SPIO (M-SPIO), ultrasmall SPIO (USPIO), and mannan-coated USPIO (M-USPIO).

Materials and methods

All experimental protocols were approved by our institutional animal experimentation committee. We intravenously injected 12 Watanabe heritable hyperlipidemic rabbits with one of the 4 types of SPIO (0.8 mmol Fe/kg). Two other rabbits served as the control. The rabbits underwent in vivo contrast-enhanced magnetic resonance angiography (MRA) before- and 5 days after these injections; excised aortae were subjected to in vitro MRI. In the in vivo and in vitro studies we assessed the signal intensity of the vessels at identical regions of interest (ROI) and calculated the signal-to-noise ratio (SNR). For histologic assessment we evaluated the iron-positive regions in Prussian blue-stained specimens.

Results

There were significant differences in iron-positive regions where M-USPIO > USPIO, M-SPIO > SPIO, USPIO > SPIO (p < 0.05) but not between M-USPIO and M-SPIO. The difference between the pre- and post-injection SNR was significantly greater in rabbits treated with M-USPIO than USPIO and in rabbits injected with M-SPIO than SPIO (p < 0.05). On in vitro MRI scans SNR tended to be lower in M-USPIO- and M-SPIO- than USPIO- and SPIO-treated rabbits (p < 0.1).

Conclusion

Histologic and imaging analysis showed that mannan-coated SPIO and USPIO particles were taken up more readily by the atherosclerotic rabbit wall than uncoated SPIO and USPIO.     

High field magnetic resonance imaging evaluation of superparamagnetic iron oxide nanoparticles in a permanent rat myocardial infarction

RATIONALE AND OBJECTIVES: The purpose of this study was to evaluate superparamagnetic iron oxide (SPIO) nanoparticles to discriminate infarcted from normal tissue after myocardial infarction using high field MR imaging (7 tesla).MATERIALS AND METHODS: Permanent myocardial infarction was induced in rats. SPIO nanoparticles (1 mg Fe/kg) were assessed with T1-weighted gradient echo sequence to visualize the myocardial infarction 48 hours after ligature (n = 6). Furthermore, MR Imaging was performed using a T2-weighted RARE sequence and nanoparticles were injected (5 or 10 mg Fe/kg) on 36 rats 5, 24 or 48 hours after infarction. RESULTS: No changes in contrast between normal and infarcted myocardium was observed after nanoparticle injection on T1-weighted images. However, nanoparticles induced a significant contrast increase between normal and infarcted myocardium on T2-weighted images whatever the delay between infarction and imaging (2.99 +/- 1.66 preinjection vs. 7.82 +/- 1.96 after SPIO injection at a dose of 5 mg Fe/kg 5 hours postinfarction, P = 0.0001). CONCLUSIONS: Nanoparticle injection made it possible to discriminate normal from infarcted myocardium on T2-weighted images. However, the high magnetic field prevented the visualization of the T1 effect of SPIO nanoparticles.     

Characteristics of ultrasmall superparamagnetic iron oxides in patients with brain tumors

OBJECTIVE: The aim of this study was to evaluate the characteristics of an ultrasmall superparamagnetic iron oxides (USPIO) agent in patients with brain tumors and to correlate changes on MRI with histopathologic data collected systematically in all patients. SUBJECTS AND METHODS: Nine patients with brain tumors were imaged before and 24 hr after administration of a USPIO at a dose of 2.6 mg Fe/kg. Analysis of MR images included qualitative and quantitative comparison of the USPIO and gadolinium enhancement of brain tumors. Brain surgery was performed 25-112 hr after administration of the USPIO. The histopathologic workup included iron histochemistry with diaminobenzidine (DAB)-enhanced Perls stain. RESULTS: In seven of nine patients, USPIO-related changes of signal intensity were observed in gadolinium-enhancing brain tumors on T1- and T2*-weighted sequences. The difference in signal intensity on T1-weighted USPIO series was 40.1% +/- 26.7% (mean +/- SD). On T2*-weighted USPIO series, the difference in signal intensity was -33.1% +/- 18.4% in solid tumor parts. Areas of suspected radiation necrosis did not enhance in three patients with prior radiation therapy. Iron histochemistry revealed the presence of iron deposits in macrophages in two patients. CONCLUSION: USPIO agents will not replace gadolinium in the workup of patients with brain tumors. Our findings suggest that USPIO agents seem to offer complementary information and may help to differentiate between brain tumors and areas of radiation necrosis. Signal intensity changes on T2*-weighted images might be related to the blood pool properties of the agent, possibly reflecting steady-state susceptibility effects.     

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.     

Improved delineation of human brain tumors on MR images using a long-circulating, superparamagnetic iron oxide agent

The purpose of this study was to corroborate experimental findings that long-circulating, superparamagnetic iron oxide contrast agents accumulate at the margins of human brain tumors, thereby improving their delineation on magnetic resonance (MR) images. This limited clinical study examined a total of four patients with brain tumors (three with primary gliomas and one with metastatic melanoma; n = 8 lesions) who were given a pharmaceutical formulation of a superparamagnetic, ultra-small-particulate iron oxide (USPIO, intravenous dose of 1.1 mg Fe/kg). The agent has a characteristically long plasma half-life and is currently undergoing Phase III clinical trials for liver disease (AMI-227, Advanced Magnetics, Cambridge, MA). MR (conventional spin-echo and gradient-echo) images of the brain were obtained before and 12, 24, and/or 36 hours after administration of the agent, with follow-up several weeks later. Twelve to 36 hours after IV administration of the USPIO, both primary and metastatic brain tumors showed readily detectable increases in signal intensity on T1-weighted spin-echo images. Unlike the pattern of enhancement with a gadolinium (Gd) chelate, which occurred immediately and decreased within hours, that with the USPIO occurred gradually, with a peak at 24 hours, and decreased over several days. Whereas the enhancing tumor margin with the Gd chelate blurred with time due to diffusion of the agent, the margin with the USPIO remained sharp, presumably due to the much lower diffusion coefficient (large size) of the particles and partly because of local endocytosis by tumor cells. Compared with Gd chelates, long-circulating, superparamagnetic iron oxide contrast agents can provide prolonged delineation of the margins of human brain tumors on MR images, which has implications for the targeting of diagnostic biopsies and the planning of surgical resections.     

Cell tagging with clinically approved iron oxides: feasibility and effect of lipofection, particle size, and surface coating on labeling efficiency

PURPOSE: To evaluate the effect of lipofection, particle size, and surface coating on labeling efficiency of mammalian cells with superparamagnetic iron oxides (SPIOs). MATERIALS AND METHODS: Institutional Review Board approval was not required. Different human cell lines (lung and breast cancer, fibrosarcoma, leukocytes) were tagged by using carboxydextran-coated SPIOs of various hydrodynamic diameters (17-65 nm) and a dextran-coated iron oxide (150 nm). Cells were incubated with increasing concentrations of iron (0.01-1.00 mg of iron [Fe] per milliliter), including or excluding a transfection medium (TM). Cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic emission spectroscopy. Cell visibility was assessed with gradient- and spin-echo magnetic resonance (MR) imaging. Effects of iron concentration in the medium and of lipofection on cellular SPIO uptake were analyzed with analysis of variance and two-tailed Student t test, respectively. RESULTS: Iron oxide uptake increased in a dose-dependent manner with higher iron concentrations in the medium. The TM significantly increased the iron load of cells (up to 2.6-fold, P < .05). For carboxydextran-coated SPIOs, larger particle size resulted in improved cellular uptake (65 nm, 4.37 microg +/- 0.08 Fe per 100 000 cells; 17 nm, 2.14 microg +/- 0.06 Fe per 100 000 cells; P < .05). Despite larger particle size, dextran-coated iron oxides did not differ from large carboxydextran-coated particles (150 nm, 3.81 microg +/- 0.46 Fe per 100 000 cells; 65 nm, 4.37 microg +/- 0.08 Fe per 100 000 cells; P > .05). As few as 10 000 cells could be detected with clinically available MR techniques by using this approach. CONCLUSION: Lipofection-based cell tagging is a simple method for efficient cell labeling with clinically approved iron oxide-based contrast agents. Large particle size and carboxydextran coating are preferable for cell tagging with endocytosis- and lipofection-based methods.     

In vitro characterization of two different ultrasmall iron oxide particles for magnetic resonance cell tracking

RATIONALE AND OBJECTIVES: Comparison of two different ultrasmall superparamagnetic iron oxide (USPIO) particles in terms of their intracellular cell-labeling properties of macrophages and subsequent visualization by MR imaging. MATERIALS AND METHODS: Cultures containing the macrophage cell line P-388D1 were incubated with a neutral carboxydextran-coated USPIO preparation (DDM 43/34/103) or an acidic citrate-coated USPIO (VSOP-C125). Experiments were performed in which incubation concentration and duration were varied and phagocytosis and pinocytosis suppressed by specific inhibitors. In cell culture specimens iron content was measured quantitatively and signal intensities determined by in vitro MR imaging. RESULTS: VSOP-C125 is incorporated by cells much faster than DDM 43/34/103 and produces significantly higher final intracellular iron concentrations per cell (3420 vs. 727 ng/million cells). Both preparations show similar signal-reducing effects at MR imaging relative to the Fe content per cell. Intracellular USPIO has a much lower detection threshold at MR imaging (50/80 micromol/L) than extracellular USPIO in free solution (300 micromol/L). CONCLUSIONS: Citrate-coated USPIO particles VSOP-C125 appear to have more favorable properties for magnetic labeling of macrophages than the carboxydextran-coated USPIO preparation DDM 43/34/103.     

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.