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

Figures & data

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.

Table 1 Iron quantification via colorimetric assay using KSCN and encapsulation efficiency estimation of purified Molday-Ion sMLs

Initial iron concentration for sML synthesis (mg Fe/mL)	Iron concentration of sMLs before purification (g Fe/mol POPC)	Iron concentration of sMLs after purification (g Fe/mol POPC)	Encapsulation efficiency* (%)
0.5	57.4 ± 2.6	53.1 ± 3.8	92 ± 3.3
1.0	119.3 ± 8.9	89.6 ± 4.6	75 ± 1.8
2.0	247.4 ± 19.6	205.7 ± 22.1	83 ± 10.4
3.0	343.1 ± 46.0	252.6 ± 38.4	74 ± 4.9

Notes:

* Percentage of encapsulation efficiency was calculated from the ratio of the iron concentration in sMLs before and after purification (EE% expressed as g Fe/mol POPC × 100). Values (±SD) are the mean of three experimental evaluations.

Abbreviations: EE%, encapsulation efficiency; POPC, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; sMLs, stealth magnetic liposomes.

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.

Table 2 Anisotropy values (<r>) of empty and Molday-Ion-loaded sMLs of different lipid compositions

Liposome formulation	Lipid molar ratio (mol/mol)	CFe in sMLs (mg/mL)	Anisotropy
POPC/CH/Peg	3/2/0.15	–	0.241 ± 0.003
POPC/CH/Peg/Fe	3/2/0.15	1.7	0.275* ± 0.013
POPC/CH/Peg + Fe	3/2/0.15	1.7	0.248** ± 0.002
POPC/Peg	3/0.15	–	0.184 ± 0.010
POPC/Peg/Fe	3/0.15	1.7	0.202* ± 0.004
POPC/CH	3/2	–	0.203 ± 0.003
POPC/CH/Fe	3/2	1.0	0.258* ± 0.009

Notes: Anisotropy values are presented as mean ± SD of three experiments.

* In all cases, sMLs showed a significantly increased anisotropy compared to the corresponding empty formulation (P < 0.01).

** Subsequent incubation with free USPIOs caused no significant increase in anisotropy (P > 0.01).

Abbreviations: sMLs, stealth magnetic liposomes; POPC, 1-Palmitoyl-2-oleoylsn-glycero-3-phosphocholine; CH, cholesterol; Peg, polyethylene glycol; C_Fe, iron concentration.

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.