Individually Stabilized, Superparamagnetic Nanoparticles with Controlled Shell and Size Leading to Exceptional Stealth Properties and High Relaxivities

Abstract

Superparamagnetic iron oxide nanoparticles (SPION) have received immense interest for biomedical applications, with the first clinical application as negative contrast agent in magnetic resonance imaging (MRI). However, the first generation MRI contrast agents with dextran-enwrapped, polydisperse iron oxide nanoparticle clusters are limited to imaging of the liver and spleen; this is related to their poor colloidal stability in biological media and inability to evade clearance by the reticuloendothelial system. We investigate the qualitatively different performance of a new generation of individually PEG-grafted core-shell SPION in terms of relaxivity and cell uptake and compare them to benchmark iron oxide contrast agents. These PEG-grafted SPION uniquely enable relaxivity measurements in aqueous suspension without aggregation even at 9.4 T magnetic fields due to their extraordinary colloidal stability. This allows for determination of the size-dependent scaling of relaxivity, which is shown to follow a d² dependence for identical core-shell structures. The here introduced core-shell SPION with ∼15 nm core diameter yield a higher R₂ relaxivity than previous clinically used contrast agents as well as previous generations of individually stabilized SPION. The colloidal stability extends to control over evasion of macrophage clearance and stimulated uptake by SPION functionalized with protein ligands, which is a key requirement for targeted MRI.

Keywords: core−shell; macrophage nanoparticle uptake; size-dependent relaxivity; superparamagnetic iron oxide nanoparticles; targeted magnetic resonance imaging.

Figure 1

Transmission electron micrographs of PEGylated SPION with diameters: (A) 3.3 ± 0.3 nm, (B) 8.7 ± 0.3 nm, (C) 10.6 ± 0.4 nm, and (D) 14.4 ± 0.8 nm. The electron diffraction pattern (inset in (C)) reveals the highly crystalline nature of the SPION, and the HR-TEM inset in (D) shows that also the 14.4 nm SPION are single-crystalline.

Figure 2

(A) Corrected transversal relaxation rates 1/T₂ as a function of Fe concentration measured in a 9.4 T field. SPION were dispersed in H₂O (circles) or agarose (triangles). Different diameters of core–shell SPION (3.3 nm - green, 8.7 nm - cyan, 10.6 nm - dark blue, 14.4 nm - gray) were compared to Ferucarbotran (Resovist) (black squares). All SPION are coated with NDA-PEG(5 kDa). The linear regression fits in water all had R² > 0.99. (B) Corrected transversal relaxivity R₂ at 1 mM_Fe as a function of d². R₂ shows a near perfect linear scaling with d² when the 3.3 nm SPION are excluded. The linear regression fit in water had R² > 0.99.

Figure 3

(A) DLS hydrodynamic diameter size distributions of 8.7 nm core–shell SPION with different coatings before MRI measurements, 8.7 nm PEGylated SPION after MRI measurements, and Ferucarbotran. Representative DLS autocorrelation functions are shown in Figure S7. (B) TEM micrograph of drop-casted Ferucarbotran and (C) 8.7 nm PEGylated core–shell NPs. The insets depict sketches of the NP preparations. Ferucarbotran has multiple cores distributed in a physisorbed carboxydextran matrix, whereas SPION in (C) have an individual covalently grafted PEG shell.

Figure 4

Macrophage uptake of different core–shell SPION architectures compared to Ferucarbotran. Cells were incubated with the respective SPION dispersions for 20 h, and the intracellular Fe content was measured by a Ferrozine assay. At least 3 independent experiments with 3 wells/experiment were performed for each data point. Statistical analysis was done by 1-way ANOVA test. ***p < 0.001, **p < 0.01, *p < 0.05. Ferucarbotran was taken up more than any of the core–shell preparations with high statistical significance (***p < 0.001), except for 8.7 nm-NDA-PEG-90B-Avidin (*p < 0.05), as indicated in the graph.

Figure 5

TEM micrographs of thin sections of macrophages incubated for 20 h with SPION. (A) Macrophages incubated with Ferucarbotran. The red arrows indicate an abundance of SPION incorporated in the endosome. (B) Macrophages incubated with PEGylated core–shell SPION (8.7 nm): no core–shell SPION could be found in endosomes in a large number of investigated cells. Pictures shown are representative for >20 cells in different thin sections that were examined.