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Published online before print April 21, 2005, 10.1148/radiol.2353032057
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(Radiology 2005;235:959-967.)
© RSNA, 2005

Molecular Imaging

Iron Oxide Nanoparticle–labeled Rat Smooth Muscle Cells: Cardiac MR Imaging for Cell Graft Monitoring and Quantitation1

Charlotte Rivière, MS, Frank P. Boudghène, MD, PhD, Florence Gazeau, PhD, Jacky Roger, MD, PhD, Jean-Noël Pons, MD, PhD, Jean-Pierre Laissy, MD, PhD, Eric Allaire, MD, PhD, Jean-Baptiste Michel, MD, PhD, Didier Letourneur, PhD and Jean-François Deux, MD

1 From the Laboratoire des Milieux Désordonnés et Hétérogènes (C.R., F.G.) and Laboratoire des Liquides Ioniques et Interfaces Chargées (J.R., J.N.P.), Université Pierre et Marie Curie, Paris, France; ERIT-M 0204 INSERM (F.P.B., J.P.L., D.L., J.F.D.) and U460 INSERM (J.B.M.), Universités Paris 7 et 13, INSERM Bldg, and Service de Radiologie (J.P.L.), Hôpital Bichat, 46 rue H. Huchard, 75877 Paris Cedex 18, France; Service de Radiologie, Université Paris 6, Hôpital Tenon, Paris, France (F.P.B., J.F.D.); and Centre de Recherches Chirurgicales, UFR de Médecine, Université Paris 12, Hôpital H. Mondor, Créteil, France (E.A.). Received December 18, 2003; revision requested February 20, 2004; final revision received June 22; accepted July 26. Supported by Institut National de la Santé et de la Recherche Médicale; Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche (ACI Technologies pour la Santé); Fondation Bettencourt-Schueller (Prix Coup d’Elan); Fondation de la Recherche Médicale; and Direction Générale de l’Armement. Address correspondence to J.F.D. (e-mail: jean-francois.deux@hmn.ap-hop-paris.fr).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To perform a quantitative analysis of anionic maghemite nanoparticle–labeled cells in vitro and determine the effect of labeling on signal intensity at magnetic resonance (MR) imaging.

MATERIALS AND METHODS: The study was approved by the institutional animal care and use committee at Hôpital Bichat. In vitro cell proliferation, iron content per cell, and MR signal intensity of cells were measured in agarose phantoms for 0–14 days of culture after labeling of rat smooth muscle cells with anionic maghemite nanoparticles. Next, iron oxide–labeled smooth muscle cells were injected into healthy hearts and hearts with ischemic injury in seven live Fisher rats. Ex vivo MR imaging experiments in excised hearts 2 and 48 hours after injection were performed with a 1.5-T medical imaging system by using T2-weighted gradient-echo and spin-echo sequences. Histologic sections were obtained after MR imaging. Correlation analyses between division factor of iron load and cell amplification factor and between 1/T2 and number of labeled cells or number of days in culture were performed by using linear regression.

RESULTS: Viability of smooth muscle cells was not affected by magnetic labeling. Transmission electron micrographs of cells revealed the presence of iron oxide nanoparticles in vesicles up to day 14 of culture. Intracellular iron concentration decreased in parallel with cell division (r2 = 0.99) and was correlated with MR signal intensity (r2 = 0.95). T2*-weighted MR images of excised rat hearts showed hypointense signal in myocardium at 2 and 48 hours after local injection of labeled cells. Subsequent histologic staining evidenced iron oxide nanoparticles within cells and confirmed the presence of the original cells at 2 and 48 hours after implantation.

CONCLUSION: Magnetic labeling of smooth muscle cells with anionic maghemite nanoparticles allows detection of cells with MR imaging after local transplantation in the heart.

© RSNA, 2005


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell therapy appears a promising field for the treatment of human diseases. As part of this new field, transplantation of smooth muscle cells has undergone extensive investigation in recent years as a potential therapy, mainly for repair of aneurysm (1) or myocardial ischemia (2) or for cardiac graft (3). Lack of ability to track the cell transplants, however, remains a major problem that must still be overcome to understand and optimize cell therapy. In fact, most cell transplantation techniques involve the use of histologic analysis to evaluate cell transfection, proliferation, and migration (4–6). There is therefore a need to develop noninvasive methods for visualizing transplanted cells.

Investigators in early studies used positron emission tomography to track radionuclide-labeled cells in vivo (7). Improvements in these techniques have since been used to analyzemetastatic tumor cell trafficking in vivo (8–11). While these techniques are sensitive, they lack high spatial resolution and require the use of nuclear labels. High-spatial-resolution magnetic resonance (MR) imaging performed with contrast agents can overcome this limitation and can be used to study the fate of magnetically labeled cells (12–16). Recent studies have demonstrated that single-cell resolution (17) and stem cell tracking are possible with contrast material–enhanced MR imaging (18).

Considerable advances have been made to enhance the efficiency of labeling and the contrast properties of labeled cells, but there have been few studies of labeling stability or quantitative assays for in vivo cell tracking. Questions such as the following are still in need of clear answers: What is the relationship between signal intensity and the number of labeled cells? How many cells can be detected in vivo? How is signal intensity related to cell proliferation and migration?

Dextran-free anionic magnetic nanoparticles are stable in colloidal suspension and are adsorbed through nonspecific electrostatic interactions with the membranes of most cell types, followed by spontaneous cell internalization (19). This label gives magnetic properties to the entire cell body, properties that potentially allow the detection of cells according to T2 and T2* values measured at MR imaging. Thus, the purpose of our study was to perform a quantitative analysis of iron oxide–labeled cells in vitro and to determine the effect of labeling on signal intensity at MR imaging.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Our study was approved by the animal care and use committee at Hôpital Bichat.

Anionic Magnetic Nanoparticles: Synthesis and Characterization
Stable colloidal suspensions of negatively charged maghemite nanoparticles were used. The precursor ionic ferrofluid was synthesized according to the Massart method (20) by alkalinizing an aqueous mixture of iron(II) chloride and iron(III) chloride. The resulting magnetite (Fe3O4) particles were then acidified, oxidized in maghemite ({gamma}-Fe2O3), and dispersed in water. This process resulted in an acidic ferrofluid composed of magnetic particles positively charged with nitrate counterions. Particles were then chelated (21) with meso-2,3-dimercaptosuccinic acid—HOOC-CH(SH)-CH(SH)-COOH—a process that forms strong complexes. An aqueous solution was obtained that contained thiolated maghemite nanoparticles that bore net negative surface charges mainly because of the unbound carboxylate groups (COO–). This colloidal solution is stable over a wide range of pH values (from 3 to 11) with suitable ionic strengths (<0.35 mol/L) and in various buffers. For this study, the nanoparticles were resuspended in a buffer solution that contained 0.1 mol/L HEPES by using serial ultracentrifugation. The nanoparticles consist of monocrystalline ferrimagnetic monodomains of maghemite with a crystalline diameter typically of 9 nm, a magnetic core of 8.7 nm, and a hydrodynamic diameter of 35 nm.

Longitudinal and transverse relaxivities (at 1.5 T, with a flip angle of 25°) of anionic magnetic nanoparticles dispersed in water are 10 and 357 sec–1 · mmol–1 · L for r1 and r2, respectively (22).

Ultrasmall superparamagnetic iron oxide particles coated with dextran (Sinerem; Guerbet, Aulnay-sous-Bois, France), of similar size (30 nm), were used as a reference.

Smooth Muscle Cell Cultures
Smooth muscle cells from Fisher rat thoracic aorta were isolated as reported previously (23). Cells were cultured in a mixture of RPMI 1640 and M199 media supplemented with 10% fetal calf serum, 50 U/mL penicillin, 40 µg/mL streptomycin, and 0.3 mg/mL L-glutamine (Gibco; Invitrogen, La Jolla, Calif). The presence of smooth muscle cells was confirmed by the appearance of focal overgrowth at confluence microscopy and by a positive result at immunohistochemical analysis for quantification of {alpha}-actin.

Magnetic Cell Labeling
Eighty-percent-confluent smooth muscle cells (passages 5–8) from the Fisher rats were incubated for 2 hours in a filter-sterilized (0.1-µm filter) suspension of anionic magnetic nanoparticles (0.1, 1.0, 5.0, and 10.0 mmol/L iron) in 0.1 mol/L HEPES at 37°C. During incubation, anionic magnetic nanoparticles adsorbed to the cell membrane surface by means of electrostatic interactions and were internalized by the cell through an endocytotic pathway (24). Cells were then washed and incubated for 1 hour in serum-free medium for chasing. After labeling and chasing, cells were washed in phosphate-buffered saline with pH of 7.4, treated with trypsin, washed again, and resuspended in culture medium by two authors independently (C.R., J.F.D.).

Quantification of Magnetic Labeling
Iron content in cells was quantified (C.R.) by using two methods: magnetophoresis and electron spin resonance (19,25). Briefly, magnetophoresis consists of measurement of the velocity of labeled cells attracted to a permanent magnet with a magnetic field of known configuration. Since the balance of magnetic and viscous forces was known, we were able to calculate the iron content of each visualized cell. To achieve reliable results, we repeated this measurement for 100 different cells. In electron spin resonance, the resonance spectrum of a known number (ie, 105) of labeled cells is recorded. Since the spectral area is proportional to total iron mass, the mean iron content per cell can be calculated. The two methods of measurement provide complementary results: Magnetophoresis provides a measure of the distribution of the whole iron load among the cells, while electron spin resonance provides a measure of average iron load determined from the cell population. The latter method yields a more reliable result if the distribution is broad or the iron content per cell is small.

Growth Kinetics and Iron Content of Smooth Muscle Cell Cultures
To monitor the fate of the magnetic nanoparticles during cell culture, the iron content per cell was analyzed and quantified with consensus by two authors (C.R., J.F.D.) according to the following procedure: After labeling with the magnetic nanoparticles (1.0 mmol/L suspension), cells were cultured for 0–14 days, with data for the different durations of culture being derived from independent cell cultures obtained from the same initial cell sample. On the indicated days, cells were treated with trypsin, washed, and resuspended in culture medium. The cell cultures were then divided into two parts: one part to be used for quantification of iron concentration and kinetic analysis of cell growth, and the other, for MR imaging. Viable trypan blue–stained smooth muscle cells were counted with a Mallasez chamber. Control cells, which were not labeled with anionic magnetic nanoparticles but were incubated in 0.1 mol/L HEPES solution for the same time as labeled cells, were also cultured. Cell iron load was quantified by means of magnetophoresis and electron spin resonance. The cell amplification factor (Af) and the division factor of iron mass per cell (DIm) were defined, respectively, as follows: Af = C(d = i)/ C(d = 0), where C is the number of cells at the given number of days (d) of culture; and DIm = Im(d = 0)/Im(d = i), where Im is the iron mass at the given number of days of culture.

The quantity of anionic magnetic nanoparticles released into the medium during culture was measured before the addition of trypsin to each cultured sample, when confluence was reached. To induce flocculation of the free magnetic particles, a 0.2 mol/L CaCl2 solution was added to the culture medium that had been in contact with the cells. The flocculants were collected by means of a strong magnet positioned on the bottom of the sample flask for 72 hours. The medium was then centrifuged at 1500 rpm for 10 minutes, and the pellets were collected for electron spin resonance analysis.

Qualitative Visualization of Iron Oxide–labeled Cells
At Perls Prussian blue staining (J.F.D.), labeled cells were fixed with 4% glutaraldehyde, washed with phosphate-buffered saline, incubated for 15 minutes in a solution of 2% potassium ferrocyanide (Perls Prussian blue stain) in 2% HCl, washed, and counterstained with eosin.

At transmission electron microscopy, anionic magnetic nanoparticle–labeled smooth muscle cells were fixed with 2% glutaraldehyde in 0.1 mol/L cacodylate buffer for 1 hour, washed three times with the same buffer solution, and then fixed again with 4% glutaraldehyde for 2 hours (C.R.). Samples were then dehydrated in an ethanol series (70%–100%), passaged through propylene oxide, and embedded in epoxy medium (EPON 812; Shell Chemical, San Francisco, Calif). Ultrathin sections (80 nm) were stained with uranyl acetate followed by lead citrate and were examined by using an electron microscope (JEM 1200 EX; JEOL, Croissy sur Seine, France). Corresponding micrographs were analyzed for samples cultured for 0–7 days with consensus by three authors (J.F.D., F.G., C.R.).

In Vitro MR Imaging of Labeled Cells
The MR detectability threshold and the pattern of change in MR signal intensity with cell growth were determined as follows: After culture and harvesting, the cells were fixed with incubation for 1 hour in 2% glutaraldehyde in 0.1 mol/L cacodylate buffer solution, washed three times with the same buffer solution, and stored at 4°C until use. The previously prepared pellets, which contained a known number of fixed labeled cells, were mixed with 0.5 mL of low temperature–melting 0.3% agarose heated to 40°C. The mixture was quickly homogenized and cooled to 4°C (C.R.).

Three types of control phantoms were prepared: agarose gels alone (without smooth muscle cells), agarose gels that contained dispersed unfixed labeled cells, and agarose gels that contained dispersed fixed nonlabeled cells.

In Vitro MR Image Acquisition
The contrast properties of labeled cells were investigated in agarose phantoms that contained a known number of dispersed fixed labeled cells with different durations of culture by using a clinical 1.5-T MR imager (Signa; GE Medical Systems, Milwaukee, Wis). All samples were placed in a head coil and imaged with a section thickness of 4 mm, field of view of 17.9 x 17.9 cm, imaging matrix of 256 x 256, and two signals acquired. The imaging protocol consisted of coronal spin-echo sequences (J.F.D.). The signal intensity was measured by using a homogeneous 20-mm2 circular region of interest. Proton relaxation times T1 and T2 were deduced from fitting of the experimental signal intensity values obtained with the spin-echo sequence as a function of repetition time (TR) (with TR of 25–3000 msec and with fixed echo time [TE]) and of TE (with TE of 10–600 msec and with fixed TR), respectively, by using an exponential law (22). In a similar way, T2* was deduced from fitting of the experimental signal intensity values obtained with the gradient-echo sequence as a function of TE.

T2 and T2* are well described with the following equation:

{r05jn20e01}
where Fe is the concentration of intracellular iron in the agarose gel and r2* is the transverse (inhomogeneous) relaxivity measured in seconds per millimole per liter. 1/T2* is thus directly proportional to intracellular iron concentration (22).

Animal Study
Male Fisher adult rats (n = 7) with a mean weight of 300 g were used for experiments. Animals were anesthetized with intraperitoneal pentobarbital (0.1 mL per 100 g body weight). Hearts were extracted after lateral thoracotomy. Left ventricular infarction was produced by ligation of the left coronary artery, as previously described (26), in four rats (J.B.M.). Fourteen days after ligation, 106 syngeneic anionic magnetic nanoparticle–labeled (incubated 2 hours with 1.0 mmol/L iron at 37°C, with 0 days of subsequent culture) smooth muscle cells in 0.5 mL of rat serum were injected in the infarcted area (three adjacent locations, 0.16 mL in each) after lateral thoracotomy (J.B.M.). Animals were sacrificed 2 hours (n = 2) or 48 hours (n = 2) after injection.

Three other rats without infarction received either an injection of 106 anionic magnetic nanoparticle–labeled smooth muscle cells (n = 1; three locations in left ventricular myocardium) or no injection (n = 2). Hearts were excised, rinsed in phosphate-buffered saline, fixed for 2 hours with 4% paraformaldehyde, and stored at 4°C in 15-mL tubes.

Ex Vivo MR Image Acquisition
Ex vivo MR imaging of the hearts was performed with the clinical 1.5-T MR imager by using a wrist coil with a diameter of 120 mm. T2-weighted spin-echo sequences were applied along the short axis of the heart and T2*-weighted gradient-echo sequences were applied along the long axis (J.F.D., J.P.L.) by using the following imaging parameters: for the T2-weighted spin-echo sequence, 2000/56 (TR msec/TE msec), 60-mm field of view, matrix of 256 x 256, and section thickness of 2 mm; for the T2*-weighted gradient-echo sequence, 16/2.8, 80-mm field of view, flip angle of 30°, matrix of 256 x 256, and section thickness of 2 mm.

Signal intensity in regions with hypointense signal (SAMNP) that indicated the presence of anionic maghemite nanoparticles on T2-weighted spin-echo images was measured with consensus by two authors (J.F.D., 4 years of experience with cardiac MR imaging; J.P.L., 20 years of experience with cardiac MR imaging) by using a circular 15-mm2 region of interest and the software available on the MR imager. The percentage of change in signal intensity (SPC) was normalized to the signal intensity of healthy myocardium (SHM) by using the equation SPC = [(SAMNP – SHM)/SHM] · 100.

Pathologic Analysis
After MR imaging, the hearts were embedded in paraffin. Sections with a thickness of 5 µm were sliced perpendicular to the long axis of the heart, from base to apex, and stained with eosin and with Perls Prussian blue stain (J.F.D.). Immunohistochemical analysis with staining for {alpha}-actin and macrophages was performed to determine the presence of smooth muscle cells. Identification of cells was confirmed by a pathologist (J.B.M.) with 10 years of experience in pathologic analysis of muscle.

Statistical Analysis
Quantitative results in study sample groups (with each group containing three to 15 data samples, unless otherwise stated) were expressed as the mean ± standard deviation. For cell growth capacity, linear regression analysis was performed for log(C) as a function of d, with C being the number of cells and d being the number of days of culture. Mean values for iron load per cell were fitted with the internalization model developed by Wilhelm et al (24). Correlations between the division factor of iron load and the cell amplification factor, as well as between 1/T2 or 1/T2* and the number of labeled cells or number of days in culture, were performed by using software for linear or first-order exponential decay regression analysis (Origin 6.0; Originlab, Northampton, Mass).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Uptake and Intracellular Localization of Anionic Magnetic Nanoparticles
Perls Prussian blue staining of cultured smooth muscle cells after 2 hours of incubation at 37°C in the suspension of anionic magnetic nanoparticles (1.0 mmol/L iron) showed intracytoplasmic iron inclusions as dense blue-stained vesicles (Fig 1a). In contrast, no iron was detected in cells after 2 hours of incubation in a suspension of dextran-coated iron oxide particles with the same iron concentration (Fig 1b). Transmission electron microscopy of labeled cells (Fig 2) indicated the presence of the anionic magnetic nanoparticles exclusively in polydisperse vesicles in the cytoplasm. The average size of the magnetic vesicles increased with cell division over time (from 173 nm ± 72 at day 0 to 600 nm ± 119 at day 7), as did the iron content.



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  		Figure 1a. Micrographs show uptake of iron oxide nanoparticles in cultured rat smooth muscle cells after 2 hours of incubation at 37°C with (a) anionic maghemite nanoparticles or (b) dextran-coated superparamagnetic iron oxide particles (1.0 mmol/L iron). Maghemite nanoparticles appeared as blue precipitate in cell cytoplasm with Perls Prussian blue staining (arrowheads in a). No iron uptake was detected in b. (Original magnification, x200; inset, x400).

 


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  		Figure 1b. Micrographs show uptake of iron oxide nanoparticles in cultured rat smooth muscle cells after 2 hours of incubation at 37°C with (a) anionic maghemite nanoparticles or (b) dextran-coated superparamagnetic iron oxide particles (1.0 mmol/L iron). Maghemite nanoparticles appeared as blue precipitate in cell cytoplasm with Perls Prussian blue staining (arrowheads in a). No iron uptake was detected in b. (Original magnification, x200; inset, x400).

 


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  		Figure 2a. Transmission electron micrographs obtained at days 0, 2, and 7 after cell labeling with anionic maghemite nanoparticles (with incubation for 2 hours at 37°C with 1.0 mmol/L iron, followed by 1-hour chasing). (a) Micrograph obtained at day 0 shows maghemite nanoparticles confined to endocytotic vesicles (white arrows) close to the cell membrane (black arrows). Micrographs obtained at days (b) 2 and (c) 7 after labeling show increasing size and particle density of dense vesicles (arrows) over time.

 


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  		Figure 2b. Transmission electron micrographs obtained at days 0, 2, and 7 after cell labeling with anionic maghemite nanoparticles (with incubation for 2 hours at 37°C with 1.0 mmol/L iron, followed by 1-hour chasing). (a) Micrograph obtained at day 0 shows maghemite nanoparticles confined to endocytotic vesicles (white arrows) close to the cell membrane (black arrows). Micrographs obtained at days (b) 2 and (c) 7 after labeling show increasing size and particle density of dense vesicles (arrows) over time.

 


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  		Figure 2c. Transmission electron micrographs obtained at days 0, 2, and 7 after cell labeling with anionic maghemite nanoparticles (with incubation for 2 hours at 37°C with 1.0 mmol/L iron, followed by 1-hour chasing). (a) Micrograph obtained at day 0 shows maghemite nanoparticles confined to endocytotic vesicles (white arrows) close to the cell membrane (black arrows). Micrographs obtained at days (b) 2 and (c) 7 after labeling show increasing size and particle density of dense vesicles (arrows) over time.

 
Cell Viability and Quantification of Magnetic Label
Smooth muscle cell viability in culture after labeling, as determined by absence of uptake of trypan blue stain, was greater than 94% ± 1. As shown in Figure 3, cell growth capacity was not affected by labeling with iron concentrations of 1.0 and 5.0 mmol/L, in the sense that the average daily rate of change in the cell count for labeled cells—with 1.0 mmol/L iron, log(C) = 5.85 + 0.12 · d; with 5.0 mmol/L iron, log(C) = 5.95 + 0.10 · d—was not significantly different from that in nonlabeled controls—log(C) = 5.87 + 0.13 · d (P > .05).



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  		Figure 3. Graph shows trends in growth of smooth muscle cells in culture after magnetic labeling (incubation at 37°C for 2 hours) with two extracellular iron concentrations (1.0 and 5.0 mmol/L). Proliferation after incubation in the absence (control) or presence of magnetic labeling was assessed by counting the number of viable cells at the reported days. Growth capacity was not affected in cells after magnetic labeling with 1 and 5 mmol/L iron, compared with growth capacity in controls.

 
Cellular uptake of iron oxide nanoparticles was evaluated for different extracellular iron concentrations of 0.1–10.0 mmol/L (Fig 4). We found a maximum iron load of 12 pg per cell with use of a high extracellular iron concentration (10.0 mmol/L) during 2-hour incubation.



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  		Figure 4. Graph shows averaged values of iron load per cell, obtained with magnetophoresis, as a function of extracellular iron concentration (0.1, 1.0, 5.0, and 10.0 mmol/L) after in vitro magnetic labeling (incubation at 37°C for 2 hours). Iron load per cell increased with increasing extracellular iron concentration until it plateaued at 12 pg per cell with extracellular iron concentration of 10.0 mmol/L.

 
Long-term studies were performed with an extracellular iron concentration of 1.0 mmol/L and 2 hours of incubation time. By means of magnetophoresis measurements, the distribution of iron load per cell was obtained for different durations of culture (Fig 5a). A shift toward lower values was observed in parallel with cell division. Intracellular mean iron load of 1.7 pg ± 0.12 per cell at day 0 of culture decreased continuously with cell division (DIm = 1.02 · Af) and reached the detection threshold at day 14 (Fig 5b), as the electron spin resonance spectrum of anionic magnetic nanoparticle–labeled smooth muscle cells and background reached comparability. A strong correlation (r2 = 0.99) between the cell amplification factor and the division factor of iron load was observed with electron spin resonance until day 7 of culture. After 7 days of culture, the magnetic load per cell did not decrease in parallel with cell growth.



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  		Figure 5a. Graphs show iron load distribution in smooth muscle cells after magnetic labeling (incubation at 37°C for 2 hours with iron concentration of 1.0 mmol/L) for days (D) 0-14 after magnetic labeling. (a) Stacked histogram shows iron load measured with magnetophoresis at days 0, 2, 3, 7, and 9. (b) Graph shows mean value of iron load per cell (black bars), measured with electron spin resonance, at days 0, 2, 3, 7, 9, and 14 after magnetic labeling. If the iron load per cell increased with the amplification factor, then iron mass remained constant until day 7 (iron mass conservation, white bars). Experiments were performed three times.

 


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  		Figure 5b. Graphs show iron load distribution in smooth muscle cells after magnetic labeling (incubation at 37°C for 2 hours with iron concentration of 1.0 mmol/L) for days (D) 0-14 after magnetic labeling. (a) Stacked histogram shows iron load measured with magnetophoresis at days 0, 2, 3, 7, and 9. (b) Graph shows mean value of iron load per cell (black bars), measured with electron spin resonance, at days 0, 2, 3, 7, 9, and 14 after magnetic labeling. If the iron load per cell increased with the amplification factor, then iron mass remained constant until day 7 (iron mass conservation, white bars). Experiments were performed three times.

 
Iron quantification in the culture medium indicated that from day 0 to day 14, iron release in the supernatant remained outside the threshold of electron spin resonance detection (4.5 x 10–11 mol/L). Therefore, the iron concentration that resulted from release of nanoparticles into the culture medium was less than 10–12 mol/L.

MR Imaging
In vitro MR imaging.—T2 and T2* were measured by using spin-echo and gradient-echo sequences, with one echo for each sequence, while varying the TE. In all cases, monoexponential signal intensity decreases were found that allowed nonambiguous determination of T2 and T2* values.

T1 also was determined by using spin-echo sequences while varying the TR. T1 of anionic magnetic nanoparticle–labeled smooth muscle cells appeared to be iron concentration independent (data not shown).

Relaxation rates 1/T2 and 1/T2* were plotted (Fig 6a, 6b) as a function of global iron concentration for two groups of phantoms: the cell density group, with different numbers (ie, 103–106) of labeled cells at day 0; and the proliferating cell group, with a constant number of cells (ie, 106) from day 0 to day 14 after labeling. As demonstrated in Figure 6a, 1/T2 was proportional to global iron concentration, with the same relaxivity (r2 = 95.8 mmol/[L · sec–1]) measured in both groups. 1/T2 thus was found to be directly proportional to the number of cells per milliliter in the agarose phantoms and to cell magnetic load (r2 = 0.97), as expressed in the equation 1/T2 = 0.004 + [(1.74 x 10–8) · C · mL–1]. While 1/T2* remained almost constant as a function of the number of cells at day 0, it decreased linearly with iron concentration in the proliferating cell group (Fig 6b).



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  		Figure 6a. Graphs show 1/T2 and 1/T2* values measured in agarose phantoms containing anionic maghemite nanoparticle-labeled cells after incubation for 2 hours at 37°C with 1.0 mmol/L iron. (a, b) Relaxation rates are shown as a function of global iron concentration for two groups of phantoms: the cell density group, in which phantoms contained different numbers of labeled cells (from 103 to 106) measured at day 0 after labeling (iron load, 1.7 pg per cell), and the proliferating cell group, in which phantoms contained a constant number of cells (106) measured from day 0 (iron load, 1.7 pg per cell) to day 14 (iron load, 0.002 pg per cell) after labeling. (c, d) Graphs (top) and corresponding MR images (bottom) obtained with T2-weighted spin-echo sequence (2000/120) show relaxation rates as a function of days of culture after labeling for (c) a constant number of cells (106) and (d) an increasing number of cells. In c, 1/T2 is directly proportional to days of culture (r2 = 0.95), whereas the decrease in 1/T2* with cell proliferation (r2 = 0.99) is best described by the nonlinear model. In d, the number of cells increased proportionally with the amplification factor (C = Af x 2.105), and 1/T2 was observed to change, on average, at the negligible rate of 2.94 x 10–5 msec–1/d. For 1/T2*, a slight decrease was observed with cell proliferation in a constant volume.

 


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  		Figure 6b. Graphs show 1/T2 and 1/T2* values measured in agarose phantoms containing anionic maghemite nanoparticle-labeled cells after incubation for 2 hours at 37°C with 1.0 mmol/L iron. (a, b) Relaxation rates are shown as a function of global iron concentration for two groups of phantoms: the cell density group, in which phantoms contained different numbers of labeled cells (from 103 to 106) measured at day 0 after labeling (iron load, 1.7 pg per cell), and the proliferating cell group, in which phantoms contained a constant number of cells (106) measured from day 0 (iron load, 1.7 pg per cell) to day 14 (iron load, 0.002 pg per cell) after labeling. (c, d) Graphs (top) and corresponding MR images (bottom) obtained with T2-weighted spin-echo sequence (2000/120) show relaxation rates as a function of days of culture after labeling for (c) a constant number of cells (106) and (d) an increasing number of cells. In c, 1/T2 is directly proportional to days of culture (r2 = 0.95), whereas the decrease in 1/T2* with cell proliferation (r2 = 0.99) is best described by the nonlinear model. In d, the number of cells increased proportionally with the amplification factor (C = Af x 2.105), and 1/T2 was observed to change, on average, at the negligible rate of 2.94 x 10–5 msec–1/d. For 1/T2*, a slight decrease was observed with cell proliferation in a constant volume.

 


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  		Figure 6c. Graphs show 1/T2 and 1/T2* values measured in agarose phantoms containing anionic maghemite nanoparticle-labeled cells after incubation for 2 hours at 37°C with 1.0 mmol/L iron. (a, b) Relaxation rates are shown as a function of global iron concentration for two groups of phantoms: the cell density group, in which phantoms contained different numbers of labeled cells (from 103 to 106) measured at day 0 after labeling (iron load, 1.7 pg per cell), and the proliferating cell group, in which phantoms contained a constant number of cells (106) measured from day 0 (iron load, 1.7 pg per cell) to day 14 (iron load, 0.002 pg per cell) after labeling. (c, d) Graphs (top) and corresponding MR images (bottom) obtained with T2-weighted spin-echo sequence (2000/120) show relaxation rates as a function of days of culture after labeling for (c) a constant number of cells (106) and (d) an increasing number of cells. In c, 1/T2 is directly proportional to days of culture (r2 = 0.95), whereas the decrease in 1/T2* with cell proliferation (r2 = 0.99) is best described by the nonlinear model. In d, the number of cells increased proportionally with the amplification factor (C = Af x 2.105), and 1/T2 was observed to change, on average, at the negligible rate of 2.94 x 10–5 msec–1/d. For 1/T2*, a slight decrease was observed with cell proliferation in a constant volume.

 


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  		Figure 6d. Graphs show 1/T2 and 1/T2* values measured in agarose phantoms containing anionic maghemite nanoparticle-labeled cells after incubation for 2 hours at 37°C with 1.0 mmol/L iron. (a, b) Relaxation rates are shown as a function of global iron concentration for two groups of phantoms: the cell density group, in which phantoms contained different numbers of labeled cells (from 103 to 106) measured at day 0 after labeling (iron load, 1.7 pg per cell), and the proliferating cell group, in which phantoms contained a constant number of cells (106) measured from day 0 (iron load, 1.7 pg per cell) to day 14 (iron load, 0.002 pg per cell) after labeling. (c, d) Graphs (top) and corresponding MR images (bottom) obtained with T2-weighted spin-echo sequence (2000/120) show relaxation rates as a function of days of culture after labeling for (c) a constant number of cells (106) and (d) an increasing number of cells. In c, 1/T2 is directly proportional to days of culture (r2 = 0.95), whereas the decrease in 1/T2* with cell proliferation (r2 = 0.99) is best described by the nonlinear model. In d, the number of cells increased proportionally with the amplification factor (C = Af x 2.105), and 1/T2 was observed to change, on average, at the negligible rate of 2.94 x 10–5 msec–1/d. For 1/T2*, a slight decrease was observed with cell proliferation in a constant volume.

 
As shown in Figure 6c, for a constant number of cells (ie, 106) from day 0 to day 14 in the phantoms, 1/T2 decreased linearly with the number of days of culture after magnetic labeling (r2 = 0.95) until it reached the level in control samples at day 14, as expressed in the equation 1/T2 = 0.011 – [(5.66 x 10–4) · d]. By contrast, the decrease in 1/T2* that occurred with cell proliferation (r2 = 0.99) is best described by the nonlinear model equation 1/T2* = 0.019 + [0.07 · exp(–d/4.02)]. Agarose phantoms with an increasing number of cells from day 0 to day 14, mimicking cell growth in a constant volume, demonstrated negligible changes in relaxation rate 1/T2 for all culture times (Fig 6d). Thus, if cells proliferate in a constant volume, 1/T2 remains constant because of iron conservation. For 1/T2*, a slight decrease in relaxivity was observed with cell proliferation in a constant volume for early days (days 0–3).

Fixation with glutaraldehyde had an effect on relaxivities: r2 and r2* in fixed cells were 96 and 520 sec–1 · mmol–1 · L, respectively, whereas r2 and r2* in unfixed cells were 291 and 380 sec–1 · mmol–1 · L, respectively.

In vivo model of ischemia.—Infarction of rat myocardium appeared as an area of high signal intensity in association with thinning of the lateral ventricular wall on T2-weighted spin-echo images and T2*-weighted gradient-echo images. At MR imaging after injection of labeled cells, lesions with hypointense signal were present in the apical region and left ventricular myocardium on T2-weighted images in both healthy hearts and hearts with ischemic injury (Fig 7). Lesions were observed on both spin-echo and gradient-echo images acquired at 2 and 48 hours after injection of labeled cells. Areas of signal intensity loss were better identified on T2*-weighted gradient-echo images than on T2-weighted spin-echo images. The percentages of change (decrease) in signal intensity on the spin-echo images for labeled cells were –52.1% ± 0.8 for healthy heart, –63.7% ± 0.4 for ischemic heart at 2 hours (n = 2), and –54.6% ± 4.8 for ischemic heart at 48 hours (n = 2). On long-axis T2-weighted gradient-echo images, sites of injected cells appeared as distinct areas of hypointense signal (Fig 7, B). Healthy excised hearts (n = 2) without labeled cells showed no areas of hypointense signal in the left myocardium (data not shown).



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  		Figure 7. A-C, Ex vivo MR images show sites of anionic maghemite nanoparticle-labeled cells. A, Short-axis view acquired with T2-weighted spin-echo sequence (2000/56; field of view, 60 mm; matrix, 256 x 256; section thickness, 2 mm) in healthy heart shows area of hypointense signal (arrowhead). B, Long-axis view obtained with T2*-weighted gradient-echo sequence (16/2.8; field of view, 80 mm; flip angle, 30°; matrix, 256 x 256; section thickness, 2 mm) 2 hours after injection of labeled cells in heart with ischemic injury shows three areas of hypointense signal (arrows) at injection site. C, Short-axis view obtained with same T2-weighted spin-echo sequence as in A, 48 hours after injection of labeled cells in heart with ischemic injury, shows area of hypointense signal (arrowhead) and thinning of left ventricular wall. D-F, Micrographs obtained 48 hours after cell injection demonstrate vesicles (arrowheads) in cells, D, in control heart, and, E, F, in heart with ischemic injury. (Perls Prussian blue stain; original magnification, x400 [D], x20 [E], x200 [F].) G, Immunohistochemical analysis was positive for {alpha}-actin (arrowhead) and thus confirmed the presence of injected cells at the same site as in F. (Eosin stain; original magnification, x200.)

 
Histologic sections obtained at the same levels as MR images and double stained with Perls Prussian blue stain and eosin showed blue vesicles within cells, a finding that indicated the presence of anionic magnetic nanoparticles in cells at 2 and 48 hours after injection in healthy and ischemic myocardium (Fig 7, D–F).

Immunohistochemical analysis of sections obtained in adjacent sites was negative for macrophages and positive for {alpha}-actin, findings that confirmed that the labeled cells were those originally injected (Fig 7, G).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
REFERENCES
 
The most commonly used iron oxide particles (27) are neutral dextran-coated particles that require different transfection agents, such as poly-L-lysine, to facilitate cell internalization (28). Other approaches, such as the use of magnetodendrimers (29) or TAT peptide–modified nanoparticles (17), have also been used. In this article, we report the use of anionic magnetic nanoparticles and their uptake by smooth muscle cells. The in vitro MR signal intensity of proliferating cells was studied. We also validated the in vivo implantation of smooth muscle cells in myocardium with MR imaging.

The uptake of anionic magnetic nanoparticles was reported previously for different species and different cell types (19), but not for smooth muscle cells. Transmission electron microscopy of labeled smooth muscle cells showed that anionic magnetic nanoparticles were confined within intracytoplasmic vesicles. The number of those vesicles decreased with cell proliferation, while their size and particle load increased. This labeling agent, as demonstrated with trypan blue exclusion counting and kinetic analysis of cell growth, is nontoxic, and it did not affect cell growth capacity. In contrast to anionic magnetic nanoparticles, the uptake of dextran-coated iron oxide particles (1.0 mmol/L iron) in smooth muscle cells was not detectable with Perls Prussian blue staining after 2 hours of incubation. In fact, the dextran-coated labeling agent follows a low-efficiency fluid phase endocytosis pathway and requires longer incubation times (>24 hours) for substantial iron uptake (19).

The quantitative uptake of the anionic magnetic nanoparticles measured with magnetophoresis and electron spin resonance immediately after the labeling procedure, up to 12 pg per cell, was comparable with that in other cell lines previously studied (19). This level of uptake is 100-fold that obtained with monodomain dextran-coated superparamagnetic iron oxide nanoparticles (16) used without a transfection agent. By using a short incubation time and low extracellular iron concentration, we achieved an efficient iron load without affecting cell viability. With longer incubation time or higher extracellular iron concentration, one can obtain an iron load per cell that is comparable with that obtained with engineered iron oxide particles modified with TAT peptide or encapsulated in dendrimers (17,29).

During cell proliferation, the simple shift of the monomodal distribution of iron load per cell measured with magnetophoresis is consistent with sharing of the magnetic load between daughter cells. There was actually a direct correlation between cell amplification factor and division factor of iron mass per cell for days of culture. Unit slope would indeed indicate a balanced sharing of particles between daughter cells. This finding was verified with electron spin resonance quantification for 0–7 days of culture (slope of 1.02). The absence of signal detection in the culture medium analyzed with electron spin resonance indicated that if exocytosis was involved, it remained at a very low level and would not interfere with MR signal detection for in vivo cell tracking. The effect of cell proliferation on the MR signal intensity was fully investigated with two independent measurements of cell iron load obtained with electron spin resonance and magnetophoresis.

At monitoring of transplanted cells in vivo, the MR signal intensity and corresponding T1, T2, and T2* values depend on two distinct relevant parameters: first, the density of labeled cells in a given volume, and, second, the intracellular particle load in labeled cells. Both parameters are involved in the proliferation and migration of transplanted cells within the targeted organ.

The T1 effect of magnetic labeling is minimal because of the confinement of magnetic particles in the cell (22). By contrast, 1/T2 is directly proportional both to the number of labeled cells and to the decrease in iron load per cell that occurs during proliferation after labeling. More generally, 1/T2 depends linearly on the global iron concentration and does not vary according to differences in cell density or cell proliferation. Moreover, cells growing in a constant volume do not induce variations in MR signal intensity. Hence, longitudinal relaxation does not depend on changes in particle load and confinement that occur during cell proliferation. Thus, 1/T2 appears to be a robust parameter for in vivo cell quantification, because its value is directly proportional to the number of cells. If cells are proliferating in a constant volume, signal intensity should remain at a constant level for a long time.

1/T2* is one order of magnitude larger than 1/T2, but its dependence on cell numbers and cell proliferation is less linear. Slight differences in 1/T2* can be observed between agarose phantoms with different cell densities and different iron loads per cell. These differences in the relaxation rate are consistent with a nonconstant signal intensity for proliferating cells in a constant volume. Hence, T2* may vary as a function of iron load per cell, size and particle density of intracellular vesicles, and overall spatial distribution of labeled cells. This variation accords with the characteristics of the T2*-weighted sequence, among which magnetic susceptibility dominates. Thus, even if the initial 1/T2* of the tissue under consideration, without labeled cells, is known, cell quantification based on 1/T2* measurement, although this technique is more sensitive than 1/T2 (30), must be performed with caution.

The transverse relaxivity (r2) of 291 sec–1 · mmol–1 · L measured for unfixed anionic magnetic nanoparticle–labeled smooth muscle cells is comparable with the r2 of 406 sec–1 · mmol–1 · L obtained for magnetodendrimers (29). Moreover, the r2 value for glutaraldehyde-fixed anionic magnetic nanoparticle–labeled smooth muscle cells was 96 sec–1 · mmol–1 · L. This decrease was probably due to the reduced proton diffusion in the cell after the fixation process. On the other hand, r2* was increased by the fixation process (from 380 to 520 sec–1 · mmol–1 · L for unfixed and fixed labeled smooth muscle cells, respectively). An explanation might be the presence of a more static local magnetic field within the fixed cells, with resultant increased field inhomogeneity.

In our in vivo experiments, we observed that efficient detection of magnetically labeled smooth muscle cells can be obtained with MR imaging by using these anionic nanoparticles. Transplanted magnetically labeled cells appeared on T2-weighted images as areas of hypointense signal both in healthy tissue and in ischemic myocardium. These results, which are in agreement with our histologic observations, suggest that MR imaging could be used to noninvasively assess cell transplantation and monitor cell engraftment in myocardium over time (31,32).

We detected anionic magnetic nanoparticle–labeled smooth muscle cells at MR imaging up to 48 hours after cell implantation. Kraitchman et al (31) observed hypointense signal areas in myocardium up to 3 weeks after cell engraftment by using magnetically labeled mesenchymal cells. Bulte et al (33) followed the migration of magnetically labeled rat neuronal progenitor cells in vivo up to 6 weeks in a rodent model of dysmyelination. One clinically relevant problem could be the uptake of magnetic particles by monocytes or macrophages if cell lysis occurs. This would lead to a failure in tracking the appropriate cells. Nevertheless, in our in vivo study, the colocalization of Perls Prussian blue stain and {alpha}-actin confirmed that the detected cells were the original injected cells. After 48 hours, labeled smooth muscle cells were still present in the area of injection. Longer times are needed to follow the fate of labeled smooth muscle cells, and experiments are currently under way with MR imaging for cell detection after 4 weeks.

Our study had several limitations. First, we did not achieve as high an iron load per cell as that (30 pg) obtained in another study with use of a low-generation heat-activated dendrimer as transfection agent (Superfect; Qiagen, Valencia, Calif) (28). The iron uptake that we documented, however, is generally sufficient for cell tracking with MR imaging.

Second, although we found a good correlation between 1/T2 and iron concentration in agarose gels in vitro, in vivo iron quantification in tissue by using MR imaging data was not performed, because we were able to determine the product of r2 and iron concentration by using the equation, without knowing either the r2 or the concentration of iron in labeled cells in the heart. In fact, for in vivo measurements, calibration of the MR signal intensity is required, with use of different numbers of labeled cells in the tissue of interest. To be accurate and of clinical use, this calibration should be performed ex vivo (to ensure a known number of labeled cells) and in fresh tissue.

In conclusion, anionic magnetic nanoparticles used for cell labeling demonstrated stable and long-lasting MR contrast properties, without cell toxicity, allowing detection at MR imaging of iron-loaded cells in myocardium. Quantitative in vitro analysis performed with MR imaging is an exciting first step toward the in vivo quantification of labeled cells. Future studies are required to follow cell engraftment over time and to fully correlate hypointense signal with the number of labeled cells. These results are promising for future in vivo study.

Although these particles are not yet clinically approved, their ease of use could be of interest for cell labeling and long-term in vivo cell tracking. The data presented here could be of interest for determining cell labeling conditions and MR imaging sequences.


   ACKNOWLEDGMENTS
 
The authors thank L. Louedec (INSERM U 460, Hôpital Bichat) for excellent technical assistance, S. Chillon (Hôpital Bichat) for ex vivo MR imaging experiments, J. Prud’homme and D. Glutron (Centre Inter-établissement de Résonance Magnetique [CIERM], Bicètre) for in vitro MR imaging experiments, N. Carreau (Université de Technologie de Compiègne) for transmission electron microscopy preparation and analysis, P. Smirnov (Laboratoire de Recherche en Imagerie, Paris) for helpful discussions, and J. Bittoun (CIERM, Bicètre) for experiments performed in his laboratory.


   FOOTNOTES
 
Abbreviations: TE = echo time, TR = repetition time

Authors stated no financial relationship to disclose.

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


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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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