Ultrasmall superparamagnetic iron oxide (USPIO)-based liposomes as magnetic resonance imaging probes

Abstract

Background: Magnetic liposomes (MLs) are phospholipid vesicles that encapsulate magnetic and/or paramagnetic nanoparticles. They are applied as contrast agents for magnetic resonance imaging (MRI). MLs have an advantage over free magnetic nanocores, in that various functional groups can be attached to the surface of liposomes for ligand-specific targeting. We have synthesized PEG-coated sterically-stabilized magnetic liposomes (sMLs) containing ultrasmall superparamagnetic iron oxides (USPIOs) with the aim of generating stable liposomal carriers equipped with a high payload of USPIOs for enhanced MRI contrast.

Methods: Regarding iron oxide nanoparticles, we have applied two different commercially available surface-coated USPIOs; sMLs synthesized and loaded with USPIOs were compared in terms of magnetization and colloidal stability. The average diameter size, morphology, phospholipid membrane fluidity, and the iron content of the sMLs were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), fluorescence polarization, and absorption spectroscopy, respectively. A colorimetric assay using potassium thiocyanate (KSCN) was performed to evaluate the encapsulation efficiency (EE%) to express the amount of iron enclosed into a liposome. Subsequently, MRI measurements were carried out in vitro in agarose gel phantoms to evaluate the signal enhancement on T1- and T2-weighted sequences of sMLs. To monitor the biodistribution and the clearance of the particles over time in vivo, sMLs were injected in wild type mice.

Results: DLS revealed a mean particle diameter of sMLs in the range between 100 and 200 nm, as confirmed by TEM. An effective iron oxide loading was achieved just for one type of USPIO, with an EE% between 74% and 92%, depending on the initial Fe concentration (being higher for lower amounts of Fe). MRI measurements demonstrated the applicability of these nanostructures as MRI probes.

Conclusion: Our results show that the development of sMLs is strictly dependent on the physicochemical characteristics of the nanocores. Once established, sMLs can be further modified to enable noninvasive targeted molecular imaging.

Keywords: MRI contrast agent; biodistribution; fluorescence polarization; magnetic liposomes.

Figure 1

Pictures of (A) sonicated liposomes loaded with polar-coated EMG-1500 USPIOs and (B) extruded sMLs encapsulating dextran-coated Molday-Ion USPIOs. Note: Photographs were taken a few hours after purification, and samples were kept at room temperature. Abbreviation: USPIOs, ultrasmall superparamagnetic iron oxides.

Figure 2

TEM micrographs of (A) dextran-coated Molday-Ion USPIOs (1.7 mg Fe/mL) in an aqueous buffer directly observed under the microscope; (B) sMLs loaded with Molday-Ion USPIOs at a final iron concentration of 1.7 mg Fe/mL (after extrusion and purification); and (C) extruded control liposomes without magnetite. Abbreviations: sMLs, stealth magnetic liposomes; TEM, transmission electron microscope; USPIOs, ultrasmall superparamagnetic iron oxides.

Figure 3

Saturation magnetization values (emu/cm³) of Molday-Ion sMLs compared to free Molday-Ion USPIOs, as a function of the Fe concentration (mg/mL). Note: The points on the diagram represent the mean value of three experiments (±SD). Abbreviations: sMLs, stealth magnetic liposomes; USPIOs, ultrasmall superparamagnetic iron oxides.

Figure 4

T2-weighted image (A) and T1-weighted scan (B) of either Molday-Ion sMLs (upper part in panels A and B) or EMG-1500sMLs (lower part in panels A and B) in 1% agarose gel phantoms. Notes: The final C_Fe (mg/mL) values correspond to the actual concentrations used for the MRI measurements shown here. Agarose gel and empty liposome are the control samples used as a reference. Abbreviations: sMLs, stealth magnetic liposomes; C_Fe, iron concentration; MRI, magnetic resonance imaging; agar, agarose gel; EL, empty liposome; ctrl, control.

Figure 5

Behavior of the kidneys, liver, and muscle. Notes: Panels (A-I and B-I) display the signal behavior of the kidneys; panels (A-II and B-II) display the signal behavior of the liver; and panels (A-III and B-III) display the signal behavior of the muscle over time. Abbreviations: sMLs, steath magnetic liposomes; USPIOs, ultrasmall superparamagnetic iron oxides.

Figure 6

Relative changes in relaxation times (qT1, qT2*) of muscle, liver, and kidneys after injection of clinical doses of 50 μmol Fe/Kg body weight in wild type mice. Notes: Signal changes were acquired at different time points (0 minutes, 5 minutes, 5 hours, 12 hours, 24 hours, 7 days) after administration of sMLs (A and C) and Molday- Ion USPIOs (B and D), respectively. A strong signal reduction was observed in liver and kidneys for both sMLs and USPIOs 5 minutes after sample administration. No visible change was measured in the muscles. Abbreviations: sMLs, stealth magnetic liposomes; USPIOs, ultrasmall superparamagnetic iron oxides; h, hours; d, days.

Figure 7

Quantification of iron in liver, kidneys, and blood by ICP-OES of control mice (n = 3) and injected mice 8 days after administration of sMLs and Molday-Ion USPIOs (n = 3 for each). Note: The values are means ± SD. Abbreviations: sMLs, stealth magnetic liposomes; USPIOs, ultrasmall superparamagnetic iron oxides, SD, standard deviation.