Effects of Clinically Relevant Superparamagnetic Iron Oxide Nanoparticles on Murine Primary Brain Cells

Superparamagnetic iron oxide nanoparticles (SPIOs) as contrast agents in magnetic resonance imaging (MRI) have been the subject of extensive research over the past decades. SPIOs are of characteristic sizes ranging from 1 to 100 nm in at least one dimension and are composed of iron oxide cores that are coated with different biodegradable materials. Due to their unique magnetic and physicochemical properties, the list of experimental and clinical research applications of SPIOs has further expanded. However, crucial information regarding interactions of clinically applied SPIOs with cells of the central nervous system (CNS) and potential toxic effects is still lacking. This is of particular importance because SPIOs are capable of penetrating biological barriers, such as the blood-brain barrier and blood-placenta barrier. The aim of this study was to investigate the morphology and viability as well as the cytokine and chemokine secretion profile of murine primary brain cells that were exposed to clinically relevant SPIOs of different sizes and compositions. For this purpose, I used primary cell cultures of microglia and hippocampal neurons cultured in monocultures and neuron-glia co-cultures. I exposed these primary cells to varying concentrations of two novel very small iron oxide particles (VSOPs) that have already passed clinical phase II trials, or the clinically approved SPIOs ferucarbotran or ferumoxytol for 6 and/or 24 hours, respectively. I show that SPIO accumulation by primary brain cells strongly depends on the cell type, exposure condition as well as particle type. Primary microglia strongly accumulated the smallest, citrate-coated VSOPs and the largest, carboxydextran-coated ferucarbotran but not the medium-sized, carboxymethyldextran-coated ferumoxytol verified by intense Prussian blue staining. Using immunocytochemistry, I show that SPIO accumulation causes morphological alterations from a ramified to an amoeboid shape, indicating microglial activation. Propidium iodide staining revealed that microglial viability was severely compromised, especially, when cells were exposed to high SPIO concentrations of 1.5 and 3.0 mM and incubated for 24 hours. While ferumoxytol was only moderately accumulated by microglia without significantly affecting viability, it still induced morphological alterations. Just as detected for microglia, only VSOPs and ferucarbotran, but not ferumoxytol, are accumulated by primary neurons, especially, after exposure to the highest iron concentration of 3.0 mM. However, all SPIOs, independent of the particle size, composition and the applied iron concentration severely affected the morphology of primary neurons from monocultures after 24 hours of exposure, which is revealed using Sholl analysis. Neurons cultured without glial cells show reduced number of neurites and increased numbers of degenerated cells in comparison to untreated neurons. In contrast, SPIO exposure of neurons in neuron–glia co-cultures seems to have a stimulatory effect on neurites. However, the analyses of secreted cytokines and chemokines secretion does not show any tendencies of SPIO-mediated effects. From the represented data I conclude that the effect of SPIOs on brain cells not only strongly depends on the respective nanoparticle type and concentration but also on the physiological microenvironment they are applied to