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Published online before print March 4, 2005, 10.1148/radiol.2351040094
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(Radiology 2005;235:155-161.)
© RSNA, 2005

Molecular Imaging

Cell Tagging with Clinically Approved Iron Oxides: Feasibility and Effect of Lipofection, Particle Size, and Surface Coating on Labeling Efficiency1

Lars Matuszewski, MD, Thorsten Persigehl, MD, Alexander Wall, MD, Wolfram Schwindt, MD, Bernd Tombach, MD, Manfred Fobker, MD, Christopher Poremba, MD, Wolfgang Ebert, PhD, Walter Heindel, MD and Christoph Bremer, MD

1 From the Departments of Clinical Radiology (L.M., T.P., A.W., W.S., B.T., W.H., C.B.) and Clinical Chemistry (M.F.), University Hospital Muenster, Albert-Schweitzer-Str 33, D-48129 Muenster, Germany; Department of Pathology, University of Duesseldorf, Duesseldorf, Germany (C.P.); Schering, Berlin, Germany (W.E.); and Interdisciplinary Center for Clinical Research, University of Muenster, Muenster, Germany (C.B.). Received January 26, 2004; revision requested March 11; revision received May 24; accepted June 28. Address correspondence to C.B. (e-mail: bremerc@uni-muenster.de).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
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 µg ± 0.08 Fe per 100 000 cells; 17 nm, 2.14 µg ± 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 µg ± 0.46 Fe per 100 000 cells; 65 nm, 4.37 µg ± 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.

© RSNA, 2005


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
The ability to track mammalian cells in vivo would have important implications for both research and clinical applications, such as stem cell therapies for tissue repair (1). During the past few years, authors of many studies have focused on tagging cells with magnetic resonance (MR)-detectable iron oxides, which allows for cell tracking with MR imaging offering high spatial resolution (ie, down to the submillimeter range). This approach is based on the high susceptibility and high spatial resolution of MR imaging (2–7).

Various techniques for cell tagging have been explored, ranging from simple incubation receptor targeting of iron oxides to sophisticated methods of iron oxide surface modification with membrane translocation signals such as the human immunodeficiency virus–derived tat peptide or magnetodendrimers (2,8).

However, in parallel, substantial research effort has been put into the development of a transfection medium (TM), including cationic peptides, dendrimers, polyamines, and lipids for nonviral transfection of DNA (9–11).

On the basis of observation of successful nonviral intracellular oligonucleotide transfer and reports of efficient magnetic labeling with magnetodendrimers (8), the purpose of this study was 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Contrast Media
A clinically approved carboxydextran-coated SPIO (SHU 555 A, Resovist; Schering, Berlin, Germany) and three derivatives with smaller hydrodynamic diameters (ultrasmall SPIOs, or USPIOs, L, M, and S; Schering) were provided. Preparations had an average hydrodynamic diameter of 65 nm (SHU 555 A), 46 nm (USPIO L), 21 nm (USPIO M), and 17 nm (USPIO S) as measured with photon-correlation spectroscopy. Their synthesis has been described in detail elsewhere (12). Furthermore, to evaluate the effect of particle coating on cell-tagging efficiency, a clinically available dextran-coated iron oxide with a hydrodynamic diameter of approximately 150 nm (Endorem; Guerbet Laboratories, Aulnay Sous Bois, France) was used (13).

All authors who are not employees of the supporting industry (Schering) had control of inclusion of the data and information for publication that might present a conflict of interest for the author who is an employee of Schering.

Cell Culture
Five cancer cell lines were investigated in this study. Human lung carcinoma cells (CLL-185) were cultured under generally established cell culture conditions in 5% CO2 at 37°C. The culture medium consisted of HAM F-12 medium (Biochrom, Berlin, Germany) that was supplemented with 10% fetal bovine serum (Gibco, Paisley, England), 1% penicillin-streptomycin (Invitrogen, Paisley, England), and 0.5% glutamine (Invitrogen). Human fibrosarcoma (HT1080), human mammary adenocarcinoma (DU447 and SKBR-3), and human lung carcinoma (HTB-56) cells were grown as described in detail elsewhere (14–21). All cell lines are commercially available from the American Tissue Culture Collection.

For harvesting CD14+ and CD14– leukocytes, peripheral blood mononuclear cells were prepared from buffy coats obtained from healthy donors (L.M., A.W.) by using Ficoll-Hypaque (Sigma-Aldrich, Munich, Germany) density gradient centrifugation. For dendritic cell generation, peripheral blood mononuclear cells were enriched for CD14+ leukocytes by using a magnetic cell separation technique (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions, which yielded more than 90% enriched monocyte fraction. Two of the authors (L.M., A.W.) consented to donate these blood samples. Our Institutional Review Board did not require its approval for this part of our study. All cell-handling procedures were performed by three of the authors (L.M., T.P., A.W.).

Cell Labeling
All experiments were performed in quadruplets. Confluent cells were split 1 day before labeling and were prepared separately for each iron concentration. After 24 hours, medium containing fetal bovine serum was replaced with medium free of fetal bovine serum to avoid unspecific binding of the SPIOs to serum albumin. Cells were then incubated with different iron concentrations of SPIOs, which ranged from 0.01 to 1.00 mg of iron [Fe] per milliliter, in the presence or absence of a lipophilic TM (Metafectene; Biontex Laboratories, Munich, Germany), for 24 hours. SPIOs varied in size, from 17 to 65 nm for a carboxydextran-coated SPIO and 150 nm for a dextran-coated SPIO. After incubation, the cells were washed four times in phosphate-buffered saline, were trypsinized, and were counted by using a calibrated cell-counting chamber (Neubauer; VWR, Darmstadt, Germany).

Cell viability was determined with trypan blue exclusion test. In this assay, cell viability is determined by using the vital dye trypan blue, which is excluded by live cells but accumulates in dead cells (22). The ratio of dead to live cells was calculated for each control and tagged-cell batch as follows: PV = (TCM – TBC)/TCM, where PV is percentage viability, TCM is the total cell number (total number of counted cells, 6.0–12.1 x 106 cells), and TBC is trypan blue–positive cells. Ten million cells of each cell batch were transferred into plastic tubes. The tubes were filled with phosphate-buffered saline to a total volume of 200 µL, and cell pellets were spun down at 10 000 rpm for 10 minutes. For MR measurements, all tubes were positioned in a plastic rack placed in oil suspension to minimize susceptibility artifacts. For determination of the detection threshold with MR imaging, cells were placed directly into an agarose gel phantom to eliminate phantom-related susceptibility artifacts. To assess the temporal label retention of dividing cells, a subset of CLL-185 cells (four cell batches; total number, 4 x 106 cells) was labeled with iron (1.00 mg Fe/mL in the medium containing TM) and was cultured for 4 weeks. Cellular iron content was measured with inductively coupled plasma atomic emission spectrometry at 1, 4, 8, 12, 16, 20, and 28 days (1 x 106 cells for each measurement).

Light and Electron Microscopy
To analyze cellular iron uptake with light microscopy, CLL-185 cells were cultured on glass slides in six-well plates, and cell labeling was performed as described earlier. Cells were fixed in 2.3% formaldehyde solution. Fixed cells were embedded in agarose and transferred into preprepared paraffin blocks. Sections for histologic examination were cut at a thickness of 4 µm. Sections were stained with Prussian blue stain (including 10 minutes with 5% potassium-hexacyanoferrat II) and were counterstained with nuclear fast red stain. The slides were examined with a microscope (Nikon Eclipse E600; Nikon, Duesseldorf, Germany) at magnifications from x20 to x400. Iron in the cytoplasm of the cells resulted in blue staining. Digital images were acquired by using a digital camera (DXM 1200; Nikon) and software (Lucia G; Nikon).

For electron microscopy, CLL-185 cells from the cell culture were fixed in a 1% dilution of 6% tannin acid (Sigma-Aldrich) and 25% glutaraldehyde (Sigma-Aldrich). Cells were embedded on Thermanox plates (NUNC, Wiesbaden, Germany), and gelatin capsules were filled with Epon (Carl Roth, Karlsruhe, Germany). Polymerization was performed for 48 hours at 60°C. Finally, electron microscopy (EM 109; Carl Zeiss, Oberkochen, Germany) was performed. Serial analyses were performed by using magnifications ranging from x4400 to x85 000.

Visual analysis at light and electron microscopy was performed by an experienced board-certified pathologist (C.P.).

Quantitative Determination of Intracellular Iron Uptake
Inductively coupled plasma atomic emission spectrometry is a well-established technique for elemental analysis and was used to quantitatively determine the intracellular iron uptake of CLL-185 cells. The technique is based on the measurement of the emitted light of excited iron atoms. The emitted wavelength is characteristic for the material that is investigated. By measuring the intensities of these wavelengths and comparing them with those generated with known standards, the concentrations of the different atoms (in this case iron) can be determined.

For the analysis of inductively coupled plasma atomic emission spectrometry, 1 million cells were prepared in 500 µL of phosphate-buffered saline. For measurements, 100 µL of cell suspension were dissolved in 100 µL of nitric acid and hydrogen peroxide and were diluted to 700 µL by adding distilled water. Iron counts were calculated automatically as micrograms of iron per liter of solution. Cell preparations and inductively coupled plasma atomic emission spectrometric (atomic absorption spectrophotometer model 2380; Perkin-Elmer, Ueberlingen, Germany) measurements were performed by two authors (L.M., M.F.).

MR Imaging Protocols
MR imaging was performed with a 1.5-T imager (Gyroscan Intera; Philips, Best, the Netherlands) by using a standard extremity knee coil. For imaging of cell phantoms, T2-weighted gradient-echo (repetition time msec/echo time msec of 250/13.81, 25° flip angle, 410 x 512 matrix, 1.5-mm section thickness), spin-echo (2500/100, 90° flip angle, 193 x 512 matrix, 1.5-mm section thickness), and turbo spin-echo (4000/100, 90° flip angle, echo train length of 15, 374 x 512 matrix, 1.5-mm section thickness) sequences were performed. The visibility of iron oxide–labeled cells on MR images was qualitatively assessed by two board-certified radiologists (C.B., B.T.).

Statistical Analysis
All data are presented as mean ± standard error of the mean. For statistical evaluation, the effect of increasing iron oxide concentrations in the medium and the influence of particle size on cellular iron uptake were compared by using an analysis of variance with a Bonferroni-corrected P value. Effects of lipofection were compared by using a two-tailed Student t test, in which cellular iron uptake with TM was compared with that without TM.

Moreover, the effect of iron labeling on cell viability was analyzed by using an unpaired two-tailed Student t test, in which cells incubated with iron-free medium were compared with cells incubated with high iron concentrations (1 mg Fe/mL incubation medium). For all tests, P < .05 was considered to indicate a significant difference. All calculations were performed by two authors (L.M., C.B.) using a commercially available statistics software (GraphPad Prism, version 4; GraphPad, San Diego, Calif).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Cell Viability Assay and Label Retention
The trypan blue exclusion assay showed an averaged viability of 96.1% ± 1.0 for the control cells. Even high iron concentrations in the incubation medium did not show any effect on cell viability (1.0 mg Fe/mL in the medium, 96.8% ± 2.5; P > .05).

Findings of longitudinal studies showed an exponential decrease of cellular iron content in dividing CLL-185 cells. The average cellular iron content decreased from 4.29 µg ± 0.6 Fe per 100 000 cells (100%) at day 1 to 1.09 µg ± 0.30 at day 4 (27.2% ± 4.1 of initial iron load). At day 8 after labeling, a 0.43 µg ± 0.16 Fe per 100 000 cells (10.3% ± 1.3 of initial iron load) was present within the cells. Three weeks after cell labeling, the iron content had returned to prelabeling baseline values (0.08 µg ± 0.02 Fe per 100 000 cells). The dilution of cellular iron oxide over time correlated well with the cell doubling time of the CLL-185 cells, which ranged between 30 and 40 hours.

Light and Electron Microscopy
Light microscopy revealed an intracellular uptake of iron oxides into the cytosol. Both the use of TM and the increasing concentration of iron oxides in the medium substantially increased the intracellular iron oxide load (Fig 1). Large particles were taken up more efficiently, compared with smaller iron oxides.



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  		Figure 1. Photomicrographs obtained at light microscopy of CLL-185 cells labeled with carboxydextran-coated iron particles (65 nm) in the presence of TM. Blue precipitate within the cells represents incorporated iron particles. A, Native cells and cells incubated with, B, 0.01; C, 0.10; and D, 1.00 mg Fe/mL. Note increasing intracellular iron uptake with higher doses of iron oxide in the incubation medium. (Prussian blue stain; objective magnification, x40).

 
Electron microscopic studies revealed a lysosomal deposition of iron oxides (Fig 2). In correlation with the trypan blue exclusion experiments, no toxic effects of the iron oxides could be observed at cellular and subcellular levels.



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  		Figure 2. Electron microscopic image of iron oxide-labeled CLL-185 cells, which were incubated with large (1.0 mg/mL) carboxydextran-coated iron oxides and fixed, reveals multiple cytoplasmatic vacuoles (arrows). Electron-dense inclusion represents iron oxide-loaded lysosomes. n = Nucleus. (Original magnification, x4400).

 
Atomic Emission Spectroscopy
With increasing iron load in the incubation medium, atomic emission spectroscopy revealed a dose-dependent increase of iron oxide uptake into the cells (65 nm: 1.0 mg Fe/mL, 1.69 µg ± 0.11 Fe per 100 000 cells vs 0.01 mg Fe/mL, 0.08 µg ± 0.01 Fe per 100 000 cells; P < .05; Fig 3). Moreover, lipofection significantly increased the iron load of cells up to 2.6-fold (Fig 3b) (65 nm: without lipofection, 1.69 µg ± 0.11 Fe per 100 000 cells vs with lipofection, 4.37 µg ± 0.08 Fe per 100 000 cells; P < .05). For carboxydextran-coated particles, larger particle size resulted in an improved iron oxide incorporation compared with smaller SPIOs (65 nm: 4.37 µg ± 0.07 Fe per 100 000 cells; 17 nm: 2.14 µg ± 0.06 Fe per 100 000 cells; P < .05) (Fig 3). Intracellular uptake of dextran-coated particles did not significantly differ from uptake of large carboxydextran-coated particles for both simple fluid-phase endocytosis and lipofection (Fig 3) (65 nm: 4.37 µg ± 0.07 per 100 000 cells vs 150 nm: 3.81 µg ± 0.39 per 100 000 cells; P > .05).



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  		Figure 3a. Quantitative iron determination at atomic emission spectroscopy. Graphs show cells incubated with SPIOs in (a) absence and (b) presence of TM. For carboxydextran-coated particles, {diamondsuit} = 65 nm, {blacktriangledown} = 46 nm, {blacktriangleup} = 21 nm, and * = 17 nm; for dextran-coated particles, {circ} = 150 nm. Note dose-dependent increase in iron uptake with increasing load of iron oxide in the incubation medium. Larger (65-150 nm) particles yielded significantly higher iron loads, compared with smaller (17-21 nm) iron oxides (P < .05). Incubation with a lipofection agent resulted in significant increase of iron oxide uptake of up to 2.6-fold (P < .05).

 


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  		Figure 3b. Quantitative iron determination at atomic emission spectroscopy. Graphs show cells incubated with SPIOs in (a) absence and (b) presence of TM. For carboxydextran-coated particles, {diamondsuit} = 65 nm, {blacktriangledown} = 46 nm, {blacktriangleup} = 21 nm, and * = 17 nm; for dextran-coated particles, {circ} = 150 nm. Note dose-dependent increase in iron uptake with increasing load of iron oxide in the incubation medium. Larger (65-150 nm) particles yielded significantly higher iron loads, compared with smaller (17-21 nm) iron oxides (P < .05). Incubation with a lipofection agent resulted in significant increase of iron oxide uptake of up to 2.6-fold (P < .05).

 
MR Imaging
Tagged human lung carcinoma cells (CLL-185) were readily visible with MR imaging, revealing strong susceptibility effects (Fig 4). For all MR measurements, the strongest susceptibility effects were obtained with the highest iron concentrations (1.00 mg Fe/mL) and in the presence of TM (Fig 4). The T2-weighted gradient-echo sequence revealed the highest susceptibility effects compared with T2-weighted spin-echo and turbo spin-echo sequences (Fig 4). By using the aforementioned gradient-echo sequences, fewer than 10 000 cells could be visualized in a cell phantom by using a standard knee coil at 1.5 T (Fig 5). Moreover, all other human cell lines tested could be sensitively detected with MR imaging after iron oxide incorporation (Fig 6). Further experiments confirmed an efficient cell tagging of noncancerous cells such as CD14+ and CD14– leukocytes (Fig 6).



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  		Figure 4. Dose-dependent susceptibility effects at MR imaging. Cells were incubated with increasing amounts of SHU 555 A (65 nm) in the presence (SPIO+TM) and absence (SPIO) of TM. A total of 1 x 105 tagged cells were imaged at 1.5 T by using T2-weighted (left) turbo spin-echo and (right) gradient-echo sequences. A dose-dependent increase of iron oxide-induced susceptibility effects could be observed. Note significantly higher susceptibility effects for cells incubated in the presence of TM.

 


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  		Figure 5. MR images show detection threshold of iron-labeled cells with clinically available MR techniques: A, 1 x 106; B, 5 x 105; C, 2.5 x 105; D, 1 x 105; E, 5 x 104; F, 2.5 x 104; G, 1 x 104; H, 7.5 x 103; I, 5 x 103; J, 2.5 x 103; K, 2 x 103; and L, 1 x 103 cells. To avoid phantom-related susceptibility artifacts, cells were embedded directly into an agarose gel phantom. As few as approximately 1 x 104 cells could be visualized by using this protocol.

 


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  		Figure 6. MR imaging demonstrates applicability of iron oxide cell tagging for other mammalian cancer cell lines. Native (upper row) and iron-labeled (lower row) cell lines at 1.5 T: A, CLL-185; B, SKBR-3; C, HTB-56; D, DU4475; E, HT1080; and F, CD14 leukocytes. All other tested cell lines were readily visible at MR imaging by using the established cell-tagging protocol.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell-based therapies have attracted increasing attention in recent years, and there is substantial hope that stem cell–based tissue repair will be of substantial benefit in various clinical entities, such as tissue remodeling after stroke or myocardial infarction (23,24). Efficient noninvasive cell-tracking modalities are essential for monitoring these therapeutic efforts. Moreover, cell-based drug targeting will require a means to sensitively detect cell movement within the living organism to ensure accurate drug delivery (25). Especially for basic research applications, there is an urgent need for reliable cell-tracking modalities. Multiple issues such as tumor-cell interactions, metastatic cell migration, and homing of stem cell populations could potentially be studied by using this approach. While many approaches to cell tagging with modified, and thus not clinically available, MR contrast agents have been described, there has been less reported work regarding the development and optimization of a cell-tagging protocol using clinically approved contrast agents (26–29). Thus, we conducted our study.

Conventional magnetic cell-labeling techniques, such as those used for cell sorting, rely on surface attachment of magnetic beads that range in size from several hundred nanometers to micrometers (30). However, surface tagging is not suitable since opsonization and rapid clearing of the cells by the reticuloendothelial system occurs (2). While few cell types exhibit a high rate of spontaneous iron oxide uptake, such as hepatic Kupffer cells, splenic macrophages, or activated microglia in the brain (31,32) for most cell types, particularly differentiated and nondividing cells, spontaneous particle uptake is generally low (2).

Recent labeling strategies for differentiated or nondividing cells have explored receptor-mediated internalization of modified iron oxides (dendrimer coating), as well as membrane translocation signals (human immunodeficiency virus–derived tat peptide) linked to iron oxides (2,8).

Bulte et al (33) explored the transferrin receptor as a vehicle for intracellular iron oxide delivery in oligodendrocyte progenitor cells using monocrystalline iron oxide nanoparticles labeled with OX-26 (receptor-mediated endocytosis). This strategy allowed for tracking of oligodendrocyte progenitor cells in myelin-deficient rats. The same group (8) synthesized dendrimer-encapsulated SPIOs (magnetodendrimers) for cell-labeling purposes. This surface modification allowed for an efficient endosomal cell labeling, with an iron oxide uptake of up to 9–14 pg per cell. Moreover, Lewin et al (2) successfully linked the human immunodeficiency virus–derived tat peptide to SPIO particles. The tat peptide sequence enables the virus to freely penetrate into the cells and therefore cross the cellular membrane. This modified SPIO showed a very efficient intracellular accumulation of up to 30 pg iron oxide per cell. However, iron oxides are in part deposited in the cell nucleus, which could, at least theoretically, harm cellular proteins and the DNA (27).

While these approaches seem to be effective, modified iron oxides are not yet clinically approved and, therefore, will not be available for human studies anytime soon. Our study adapted an existing, clinically approved contrast media technology for cell targeting.

Efficacy of Cell Tagging
We did observe spontaneous fluid-phase endocytosis in all dividing cell types, which resulted in a substantial uptake of iron oxides into the cells (up to 2 µg Fe per 1 x 105 cells). This is not surprising, since spontaneous uptake of iron oxides has been shown previously by Moore et al (34). However, the best results of iron labeling in this study were achieved with the use of carboxydextran-coated large SPIOs in the presence of the TM, yielding about 5 µg Fe per 100 000 cells, which is within the range of what Lewin et al (2) and Bulte et al (8) described in their study. Similar results were recently obtained by Frank et al (27), who reported successful cell tagging by using a dextran-coated iron oxide (MION-46L) and a commercially available TM. The same group recently showed that viability, growth rate, and apoptotic index are unaffected by endosomal SPIO incorporation, which is also confirmed by our data (35).

Hoehn et al (26) applied a comparable cell-tagging technique for monitoring of stem cell migration in an experimental stroke model. Daldrup-Link et al (29) investigated iron oxides, which are either clinically approved or in clinical testing for cell tagging. They looked at receptor-mediated endocytosis, fluid-phase endocytosis, and transfection methods by using liposomes of different contrast agents in hematopoetic progenitor cells. Interestingly, their labeling efficacy was on the order of one magnitude lower than the findings we observed in our study and resulted in a lower detection threshold of the cells (11 x 105 cells at 1.5 T). This may be related, in part, to the different endocytotic activity and differing cell size of hematopoetic cells, compared with the mammalian tumor cells in our study. In this context, Moore et al (34) were able to show that spontaneous cellular iron oxide uptake correlates well with the proliferation rates of different cell types.

Size Dependency of Particle Uptake
Endocytosis is the major route of lipoplex entry into cultured cells (36–38), and it is well known that endocytosis is affected by particle size (39,40).

The results of our experimental study show a clear dependency of particle size and cellular uptake, with significantly higher cellular uptake of larger than of smaller carboxydextran-coated iron oxide particles. These findings are in line with in vitro results of Ross and Hui (41), who looked at lipofection-based cellular uptake of fluorescently labeled latex beads. In that study, particles ranging in size from 35 to 2200 nm showed a linear increase of cellular particle uptake with increasing particle size. Moreover, Ross and Hui (41) showed that the size of liposomes encapsulating latex beads is correlated with lipofection efficacy regardless of liposome type.

It might, therefore, be speculated that a further increase of iron oxide particle size should be useful for cell-tagging MR studies because of an improved cellular uptake and an enhanced T2 effects due to an increase in r2/r1 ratio with increasing particle size (12). Authors of future studies should, therefore, evaluate if increasing particle size will further enhance the sensitivity of MR imaging for cell detection.

Surface Coating
For intravenous imaging applications, the biodistribution of iron oxide particles is known to be influenced not only by the size of the particles but also by the surface coating. Uptake and pharmacokinetics of different iron oxide particles have been studied extensively during the past few years (41–43). It could be speculated that since lipofection results in liposome coverage of iron oxides, the particle surface structure should be efficiently shielded, and, therefore, the particle coating will be of minor importance for cell-tagging efficacy. However, we achieved maximum cellular iron loading for both fluid-phase endocytosis and lipofection by using large (65 nm) carboxydextran-coated particles. Although substantially larger (150 nm), dextran-coated iron oxide particles revealed virtually identical or lower cellular iron uptake. This is in contradistinction to the observations made in this study and in the previously mentioned investigation by Ross and Hui (41), which showed an increase of cellular particle uptake with increasing particle size. Therefore, for high loading of cells with iron oxides, which is a prerequisite for sensitive in vivo cell tracking, carboxydextran coating seems superior to dextran coating. However, we can only provide indirect evidence to prove this fact. Further studies should therefore focus on a systematic investigation of iron oxide surface coating and entry into cells of liposomes encapsulating latex beads.

In summary, the results of this study demonstrate the feasibility of efficient magnetic cell tagging with simple lipofection techniques by using clinically approved iron oxide particles. Large particle size and carboxydextran coating are preferable for both fluid-phase endocytosis and lipofection of cells with iron oxides. Future studies should focus on applying this cell-labeling technique as an off-label use of iron oxide particles in clinical cell-tracking studies.

Cell-tagging protocols are necessary for monitoring stem cells therapies, which are currently under development for tissue repair in patients with myocardial infarction, ischemic stroke, or neurodegenerative disorders (31,33,44). Moreover, cell-based anticancer therapies will require sensitive means to detect antigen-specific cytotoxic leukocytes (45,46). Finally, visualization of inflammatory cells may be a substantial help for localization and grading of inflammatory diseases (eg, autoimmune disease).


   FOOTNOTES
 
Abbreviations: SPIO = superparamagnetic iron oxide, TM = transfection medium

See Materials and Methods for pertinent disclosures.

Author contributions: Guarantors of integrity of entire study, L.M., C.B.; study concepts, L.M., C.B.; study design, L.M., W.S., B.T., W.E., C.B.; literature research, L.M., T.P., A.W., C.B.; experimental studies, L.M., T.P., A.W., M.F., C.P., C.B.; data acquisition, L.M., T.P., M.F., C.P., A.W., C.B.; data analysis/interpretation, L.M., B.T., W.E., C.B.; statistical analysis, L.M., C.B.; manuscript preparation and editing, L.M., C.B.; manuscript definition of intellectual content and revision/review, L.M., W.E., B.T., W.H., C.B.; manuscript final version approval, L.M., W.H., C.B.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 

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