Comparative in vitro and in vivo Evaluation of Different Iron Oxide-Based Contrast Agents to Promote Clinical Translation in Compliance with Patient Safety

Figures & data

Table 1 Summary of MRI Acquisition Parameters for in vivo Imaging

Type of Scan	Repetition Time/Echo Time (ms/ms)	vField of View (mm³)	Resolution (mm³)	Gap Between Slices (mm)	Number of Animals
T1w TSE	630.8/20	70 × 70 × 57	0.3125 × 0.3125 × 1.5	0.8	3
T2w TSE SPAIR	2000/40	70 × 70 × 54	0.3125 × 0.3125 × 1.5	0.15	3
T2*w FFE	11.173/6.638	70 × 70 × 64.5	0.49 × 0.49 × 1.5	–	3

Abbreviations: T_iw (i=1, 2 or 2*), T_i-weighted, TSE, turbo spin echo, SPAIR, spectral attenuated inversion recovery, FFE, fast field echo.

Table 2 Summary and Comparison of the Contrast Agent Formulations’ Different Physicochemical Properties

Property	SPION^Dex	Ferucarbotran	Ferumoxytol
Hydrodynamic size (nm)	35 ± 0.1	59 ± 0.1	38 ± 0.2
Polydispersity index (a.u.)	0.129 ± 0.005	0.180 ± 0.013	0.210 ± 0.001
ζ-Potential (mV) at pH 7.4	−4 ± 0.3	−35 ± 0.4	−40 ± 0.5
Magnetic volume susceptibility normalized to iron concentration (mL/mg)	9.0 × 10^−4 ± 4 × 10^−6	7.7 × 10^−3 ± 9 × 10^−7	1.5 × 10^−3 ± 3 × 10^−7
r1 (s^−1mM^−1)	6 ± 0.4	1.4 ± 0.1	6 ± 0.4
r2 (s^−1mM^−1)	66 ± 1.6	30 ± 2.0	67 ± 1.7
r2* (s^−1mM^−1)	101 ± 3.0	86 ± 5.8	98 ± 4.0

Note: Mean ± SD of n=3 experiments are shown.

Abbreviations: r_i, relaxivity of T_i-weighted sequences (i= 1, 2 or 2*), SPION^Dex, dextran-coated superparamagnetic iron oxide nanoparticles, SD, standard deviation.

Figure 1 Comparison of physicochemical properties of the particles. (A) Intensity-weighted hydrodynamic size distribution. (B) -potential distribution at pH 7.4. 3 T MRI derived T₁ (C), T₂ (D) and T₂* (E) relaxation rates as a function of iron concentration. The relaxivity r is derived from the slope of the corresponding linear fit.

Notes: For (A and B): representative result of one experiment. For (C–E): mean ± SD of n=3 experiments are shown.

Abbreviations: R_i, relaxation rate of T_i-weighted sequences (i= 1, 2 or 2*), SPION^Dex, dextran-coated superparamagnetic iron oxide nanoparticles, SD, standard deviation.

Figure 2 Cytotoxicity of nanoparticles. (A) Viability of Jurkat cells after treatment with nanoparticles for 24 h determined by AxV-PI staining. (B) Cellular nanoparticle uptake determined by FACS side scatter intensity. (C) Reactive oxygen species formation in cells measured by DCFH fluorescence. (D) Effect of contrast agents on intrinsic blood coagulation pathway. (E) Activation of thrombocytes. (F) Formation of neutrophil extracellular traps.

Notes: Asterisks mark the significance levels after passed normality test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). For (A–C) mean ± SD of n=5 experiments are shown. For (D–F) mean ± SD of n=3 experiments are shown.

Abbreviations: SPION^Dex, dextran-coated superparamagnetic iron oxide nanoparticles, a.u., arbitrary units, DCFH, 2’-7ʹdichlorofluorescin, aPTT, activated partial thromboplastin time, SD, standard deviation.

Figure 3 Contrast agent-induced complement activation. (A) Determination of the iC3b split product, in human-derived platelet-poor plasma (n=3). Relative hemodynamic changes as indication of in vivo complement activation in a porcine model after administration of SPION^Dex (B), ferucarbotran (C), and ferumoxytol (D) at an iron concentration of 5 mg/kg. The latter resulted in anaphylaxis and requiring subsequent cardiopulmonary resuscitation. (E) Comparisons of maximum pulmonary artery pressure after contrast agent application (data for both doses, 0.5 and 5.0 mg Fe/kg, are pooled). (F) Normalized thromboxane B2 levels in pigs acquired from blood samples collected during application. (G) Example image of flushes appearing in the abdominal region after ferumoxytol application.

Notes: Asterisks mark the significance levels after passed normality test (***p < 0.01, ***p < 0.001, ****p < 0.0001). For (A): mean ± SD of n=3 experiments are shown. For (B–D): representative result of one experiment are shown. For (E and F): mean ± SD of n=4 experiments are shown (data for both doses, 0.5 and 5.0 mg Fe/kg, are pooled).

Abbreviations: SPION^Dex, dextran-coated superparamagnetic iron oxide nanoparticles, iC3b, protein fragment that is part of the complement system, CVF, cobra venom factor, PAP_max, maximum pulmonary artery pressure, TXB2, thromboxane B2, SD, standard deviation.

Figure 4 MRI experiments in rats. (A) Exemplary transversal T₂*-weighted MRI of a rat’s liver before and at specific time points after administration of contrast agents at an iron dose of 2.6 mg Fe /kg. Qualitative evaluation of liver uptake and elimination of contrast agents from the liver based on T₁-weighted (B), T₂-weighted (C), and T₂*-weighted (D) scans (n=3).

Notes: Asterisks mark the significance levels after passed normality test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Mean ± SD of n=3 experiments are shown.

Abbreviations: SPION^Dex, dextran-coated superparamagnetic iron oxide nanoparticles, SD, standard deviation.