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

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).

View larger version (158K): [in a new window]   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).

View larger version (184K): [in a new window]   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).

View larger version (98K): [in a new window]   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.

View larger version (113K): [in a new window]   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.

View larger version (107K): [in a new window]   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.

View larger version (28K): [in a new window]   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.

View larger version (20K): [in a new window]   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.

View larger version (34K): [in a new window]   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.

View larger version (24K): [in a new window]   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.

View larger version (21K): [in a new window]   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.

View larger version (23K): [in a new window]   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.

View larger version (29K): [in a new window]   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.

View larger version (32K): [in a new window]   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.

View larger version (93K): [in a new window]   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 -actin (arrowhead) and thus confirmed the presence of injected cells at the same site as in F. (Eosin stain; original magnification, x200.)