Lysosomal degradation of the carboxydextran shell of coated superparamagnetic iron oxide nanoparticles and the fate of professional phagocytes
Introduction
Kupffer cells are resident liver macrophages, which represent ∼50% of all macrophages of the body [1]. Macrophages such as Kupffer cells are crucial for the coordination of immune responses, elimination of pathogens, and control of tissue homeostasis. They produce a range of cytokines and mediators necessary for the orchestration of innate and adaptive immune responses. Kupffer cells are involved in the regulation of hepatocyte proliferation and metabolism. Hence, dysregulation of these processes can be of pathogenic relevance [1].
Macrophages are also crucial for the recycling of most of the iron content of the body. Due to high-level expression of specific storage proteins, macrophages can sustain a relatively high iron load [2]. Nevertheless, during inflammation, TNF-α and other proinflammatory cytokines may facilitate accumulation of iron that may impair the viability of macrophages and their neighboring cells [2], [3].
Superparamagnetic nanoparticles are increasingly used as contrast agents for diagnostic magnetic resonance imaging (MRI) of focal hepatic lesions and for the identification of molecular targets in various disease states [4], [5]. Most contrast agents remain extracellular, such as gadolinium complexes, which have a relatively short residence time in the vascular system. They have no tissue specificity and are excreted through the kidneys. More recently, agents have been introduced that possess longer residence times, mainly because they are recruited intracellularly, and therefore allow extended imaging procedures [6].
Nanosized superparamagnetic iron oxide contrast agents, also called SPIO (“small particle iron oxides” or “superparamagnetic iron oxides”), remain in the circulation for extended periods of time depending on particle size and surface coating. Finally, however, these particles are taken up by the reticuloendothelial system, which consists mainly of macrophages [7]. SPIO particles are currently in use as contrast agents for diagnostic MRI of the liver and bowel [6]. The SPIO ferucarbotran (SHU 555A) has been used for diagnostic liver imaging of transplantation patients, patients with liver cirrhosis, metastases and suspected hepatocellular carcinoma [8]. This agent appears to be well suited for the diagnostic differentiation between benign versus malignant liver lesions as well as for proving or excluding multifocal liver lesions [8], [9].
Ferucarbotran has been considered a safe and well tolerated agent [8]. Most clinical studies reported only mild adverse reactions shortly after ferucarbotran administration [10], [11], [12]. However, gadolinium-containing contrast agents were initially also considered to be safe until toxicity that manifested itself after 2–4 weeks or even later was finally recognized [13]. Therefore, toxicological studies limited to short periods of time after injection should be regarded with great caution.
So far, delayed effects of SPIO nanoparticles on professional phagocytes have not been investigated thoroughly. Therefore, in this study we have aimed at investigating the long-term effects of the SPIO ferucarbotran on Kupffer cell viability in mice in vivo in comparison to its effects on human macrophages in vitro.
Section snippets
Materials
The SPIO ferucarbotran (SHU 555A, Resovist™) was obtained from the pharmacy of the University Hospital and has previously been characterized with a Zetasizer Nano in terms of average size, electrophoretic mobility, zeta potential, and surface charge density [14].
Animal experiments
Ferucarbotran was injected in the tail vein of NMRI mice (28–33 g) (Janvier Laboratories) as a bolus of 10 μmol Fe/kg suspended in 100 μl 0.9% NaCl. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) (Calbiochem) dissolved in DMSO and
MRI analysis of SPIO distribution in mice
Intravenous bolus injection of the recommended dose of ferucarbotran nanoparticles (10 μmol Fe/kg) resulted in a strong signal loss in the liver tissue of all mice analyzed by MRI (Fig. 1A). By contrast, there were no changes in the signal intensity of the control muscle tissue. The signal reduction in the liver was maximal 1 h after injection and decreased continuously over eight days; it still remained significant, however, even after three weeks (Fig. 1A and B) clearly indicating long-term
Discussion
As macrophages are equipped with all necessary tools to sense, internalize and digest particulate matter, it is not surprising that the hepatic macrophages, the Kupffer cells, take up a significant portion of injected nanosized particles [25].
Here we have shown that 5 days after injection of a diagnostically relevant dose of the SPIO ferucarbotran [8], tissue levels of iron remain significantly elevated in murine liver and kidneys as compared to control animals. The observed increases in
Conclusions
Professional phagocytes deposit the carboxydextran-coated SPIO ferucarbotran in lysosomal vesicles thereby exposing the nanoparticles to lysosomal α-glucosidase that effectively degrades the carboxydextran shell. This leads to liberation of the iron oxide core that catalyzes ROS generation, JNK activation and subsequent apoptosis, and finally Kupffer cell depletion in vivo. Therefore, the cytotoxicity of SPIO-based contrast agents specifically for professional phagocytes should be considered,
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Priority Program SPP1313 and the Center for Functional Nanostructures (CFN).
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