Biodistribution, kinetics, and biological fate of SPION microbubbles in the rat

Figures & data

Figure 1 Properties of SPION microbubbles. SPION microbubbles seen under light microscopy (image size 550 μm × 550 μm) using the fluorescein isothiocyanate channel (A) and in bright-field microscopy setting (B). Two microbubbles seen on electron microscopy (C) and crystal lattice parameters of SPION (D) using field emission transmission electron microscopy.

Abbreviation: SPION, superparamagnetic iron oxide.

Figure 2 Flow cytometric analysis of SPION microbubbles. FSC shows size distribution (A and C) and SSC provides information about the surface (B and C), indicating a narrow size distribution and homogeneous surface. The different fluorescent lasers [FL1 (D) 536 ± 40 nm; FL2 (E) 590 ± 50 nm; FL3 (F) 675 nm] show that the dye is homogenously distributed among the SPION microbubbles.

Abbreviations: FSC, forward scattering; SSC, sideward scattering; SPION, superparamagnetic iron oxide.

Figure 3 Polarizing properties of SPION microbubbles. Single and clustered SPION microbubbles in pulmonary vasculature and macrophages (10 minutes post injection) show birefringent walls when viewed using polarized light (A). At 3 weeks post injection, a decrease in birefringence is shown in phagocytosed SPION microbubbles [arrow, (B)]. The size bar represents 10 μm.

Abbreviation: SPION, superparamagnetic iron oxide.

Figure 4 Dynamic magnetic resonance imaging of a rat after injection of SPION microbubbles. Magnetic resonance signal intensity changed mostly in the liver over time from 10 minutes to 6 weeks compared with Pre-injection. (A) Pre-injection and at (B) 10 minutes, (C) one hour, (D) 24 hours, (E) 48 hours, (F) one week, (G) 2 weeks, (H) 4 weeks, and (I) 6 weeks post injection.

Abbreviation: SPION, superparamagnetic iron oxide.

Figure 5 Pharmacokinetic model of SPION microbubbles. Relaxation times (R2*; s⁻¹) of SPION microbubbles in the liver (as indicator for concentrations) versus time (hours) were fitted to a one-compartment open model. The elimination half-life was calculated from the slope.

Abbreviation: SPION, superparamagnetic iron oxide.

Figure 6 Histology of the lungs, liver, and spleen of a rat post injection of SPION microbubbles. Abundant intravascular, fibrin-covered microbubbles in lung at 10 minutes (A) and 24 hours (B). A decreased number of phagocytosed microbubbles were observed at 2 weeks (C). Iron pigment becomes apparent in macrophage cytoplasm after 4 weeks (D). In the liver, rare microbubbles are found in the vicinity of Kupffer cells at 10 minutes post injection (E), and increasingly phagocytosed at 24 hours (F). Phagocytosed microbubbles were abundant at 2 weeks post injection (G) and decreased 4 weeks after injection (H). In the spleen, 10 minutes after injection, microbubbles are found in red pulp and marginal zone (I). From 24 hours after injection and onwards, the microbubbles are associated with marginal zone macrophages and rare in red pulp (J). (K) at 2 weeks and (L) at 4 weeks. The size bar represents 10 μm. The arrows show the microbubbles in the different tissues.

Abbreviation: SPION, superparamagnetic iron oxide.

Figure 7 Distribution of iron after injection of SPION microbubbles in rat lungs, liver, and spleen at various time points visualized by Perl’s Prussian blue staining. SPION microbubbles/iron at 10 minutes, one week, and 6 weeks post injection in (A–C) the lungs, (D–F) liver, and (G–I) spleen. The size bar represents 25 μm.

Abbreviation: SPION, superparamagnetic iron oxide.

Figure 8 Digital quantification of the number of Perl’s Prussian blue-stained SPION microbubbles in the lungs, liver, and spleen over time.

Abbreviations: SPION, superparamagnetic iron oxide; MBs, microbubbles; mins, minutes; h, hours; w, weeks.

Figure 9 The amount of iron in the spleen of 12-week-old and 25-week-old control rats compared with a 25-week-old rat injected with SPION microbubbles as visualized using Perl’s Prussian blue staining. (A) The spleen of a 12-week-old control rat (untreated) shows a limited amount of iron, while (B) in the 25-week-old control rat, the amount of iron was increased. (C) Shows the amount of iron found in the spleen of the 25-week-old rat injected 6 weeks previously with SPION microbubbles, that was considerably higher compared with the untreated control.

Abbreviation: SPION, superparamagnetic iron oxide.

Figure 10 Intracellular localization of fluorescent SPION microbubbles inside labeled macrophages. A primary antibody against CD68 (red) confirms phagocytosis of the microbubbles (green) by macrophages in the lungs. The nuclei were stained blue using DAPI. The size bar represents 25 μm. The upper left part of the image is higher magnification with the size bar of 10 μm.

Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; SPION, superparamagnetic iron oxide.

Figure 11 Protracted pulmonary pathology and example of glomerular SPION microbubbles. (A) Macrophages engulfing SPION microbubbles in a mural intravascular thrombus associated with neutrophils in the lung of one rat 24 hours post injection and (B) localized chronic inflammation characterized by accumulated macrophages, fewer neutrophils, and marked fibrosis in the lung of a rat, 4 weeks post injection of SPION microbubbles. (C) Renal cortex showing occasional SPION microbubbles in the glomerular vascular tuft. Size bars represent 50, 10, and 10 μm, respectively.

Abbreviation: SPION, superparamagnetic iron oxide.