Charlotte Petters

Handling of Iron Oxide and Silver Nanoparticles by Astrocytes

Metal-containing nanoparticles (NPs) are currently used for various biomedical applications. Since such NPs are able to enter the brain, the cells of this organ have to deal with NPs and with NP-derived metal ions. In brain, astrocytes are considered to play a key function in regulating metal homeostasis and in protecting other brain cells against metal toxicity. Thus, among the different types of brain cells, especially astrocytes are of interest regarding the uptake and the handling of metal-containing NPs. This article summarizes the current knowledge on the consequences of an exposure of astrocytes to NPs. Special focus will be given to magnetic iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs), since the biocompatibility of these NPs has been studied for astrocytes in detail. Cultured astrocytes efficiently accumulate IONPs and AgNPs in a time-, concentration- and temperature-dependent manner by endocytotic processes. Astrocytes are neither acutely damaged by the exposure to high concentrations of NPs nor by the prolonged intracellular presence of large amounts of accumulated NPs. Although metal ions are liberated from accumulated NPs, NP-derived iron and silver ions are not exported from astrocytes but are rather stored in proteins such as ferritin and metallothioneins which are synthesized in NP-treated astrocytes. The efficient accumulation of large amounts of metal-containing NPs and the upregulation of proteins that safely store NP-derived metal ions suggest that astrocytes protect the brain against the potential toxicity of metal-containing NPs.

Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells

Microglia are the phagocytotic cells of the brain that respond rapidly to alterations in brain homeostasis. Since iron oxide nanoparticles (IONPs) are used for diagnostic and therapeutic applications in the brain, the consequences of an exposure of microglial cells to IONPs are of particular interest. To address this topic we have synthesized and characterized fluorescent BODIPY®-labelled IONPs (BP-IONPs). The average hydrodynamic diameter and the ζ-potential of BP-IONPs in water were ∼65 nm and -49 mV, respectively. Both values increased after dispersion of the particles in serum containing incubation medium to ∼130 nm and -8 mV. Exposure of cultured rat microglial cells with BP-IONPs caused a time-, concentration- and temperature-dependent uptake of the particles, as demonstrated by strong increases in cellular iron contents and cellular fluorescence. Incubation for 3h with 150 and 450 μM iron as BP-IONPs increased the cellular iron content from a low basal level of ∼50 nmol iron mg(-1) to 219±52 and 481±28 nmol iron (mg protein)(-1), respectively. These conditions did not affect cell viability, but exposure to higher concentrations of BP-IONPs or for longer incubation periods severely compromised cell viability. The BP-IONP fluorescence in viable microglial cells was co-localized with lysosomes. In addition, BP-IONP accumulation was lowered by 60% in the presence of the endocytosis inhibitors 5-(N-ethyl-N-isopropyl)amiloride, tyrphostin23 and chlorpromazin. These results suggest that the rapid accumulation of BP-IONPs by microglial cells is predominantly mediated by macropinocytosis and clathrin-mediated endocytosis, which direct the accumulated particles into the lysosomal compartment.

Uptake and Metabolism of Iron Oxide Nanoparticles in Brain Cells

Magnetic iron oxide nanoparticles (IONPs) are used for various applications in biomedicine, for example as contrast agents in magnetic resonance imaging, for cell tracking and for anti-tumor treatment. However, IONPs are also known for their toxic effects on cells and tissues which are at least in part caused by iron-mediated radical formation and oxidative stress. The potential toxicity of IONPs is especially important concerning the use of IONPs for neurobiological applications as alterations in brain iron homeostasis are strongly connected with human neurodegenerative diseases. Since IONPs are able to enter the brain, potential adverse consequences of an exposure of brain cells to IONPs have to be considered. This article describes the pathways that allow IONPs to enter the brain and summarizes the current knowledge on the uptake, the metabolism and the toxicity of IONPs for the different types of brain cells in vitro and in vivo.

Bifunctional Submicron Colloidosomes Coassembled from Fluorescent and Superparamagnetic Nanoparticles

Colloidosomes are microcapsules consisting of nanoparticle shells. These microcarriers can be self-assembled from a wide range of colloidal particles with selective chemical, physical, and morphological properties and show promise for application in the field of theranostic nanomedicine. Previous studies have mainly focused on fairly large colloidosomes (>1 μm) based on a single kind of particle; however, the intrinsic building-block nature of this microcarrier has not been exploited so far for the introduction of tailored functionality at the nanoscale. We report a synthetic route based on interfacial shear rheology studies that allows the simultaneous incorporation of different nanoparticles with distinct physical properties, that is, superparamagnetic iron oxide and fluorescent silica nanoparticles, in a single submicron colloidosome. These tailor-made microcapsules can potentially be used in various biomedical applications, including magnetic hyperthermia, magnetic particle imaging, drug targeting, and bioimaging.

Lysosomal iron liberation is responsible for the vulnerability of brain microglial cells to iron oxide nanoparticles: comparison with neurons and astrocytes.

Abstract Iron oxide nanoparticles (IONPs) are used for various biomedical and neurobiological applications. Thus, detailed knowledge on the accumulation and toxic potential of IONPs for the different types of brain cells is highly warranted. Literature data suggest that microglial cells are more vulnerable towards IONP exposure than other types of brain cells. To investigate the mechanisms involved in IONP-induced microglial toxicity, we applied fluorescent dimercaptosuccinate-coated IONPs to primary cultures of microglial cells. Exposure to IONPs for 6 h caused a strong concentration-dependent increase in the microglial iron content which was accompanied by a substantial generation of reactive oxygen species (ROS) and by cell toxicity. In contrast, hardly any ROS staining and no loss in cell viability were observed for cultured primary astrocytes and neurons although these cultures accumulated similar specific amounts of IONPs than microglia. Co-localization studies with lysotracker revealed that after 6 h of incubation in microglial cells, but not in astrocytes and neurons, most IONP fluorescence was localized in lysosomes. ROS formation and toxicity in IONP-treated microglial cultures were prevented by neutralizing lysosomal pH by the application of NH4Cl or Bafilomycin A1 and by the presence of the iron chelator 2,2′-bipyridyl. These data demonstrate that rapid iron liberation from IONPs at acidic pH and iron-catalyzed ROS generation are involved in the IONP-induced toxicity of microglia and suggest that the relative resistance of astrocytes and neurons against acute IONP toxicity is a consequence of a slow mobilization of iron from IONPs in the lysosomal degradation pathway.

Accumulation of iron oxide nanoparticles by cultured primary neurons

Magnetic iron oxide nanoparticles (IONPs) are frequently used for biomedical applications. Although nanoparticles can enter the brain, little is known so far on the uptake of IONPs in neurons and on their neurotoxic potential. Hence, we applied dimercaptosuccinate (DMSA)-coated IONPs to cultured primary rat cerebellar granule neurons. These IONPs had average hydrodynamic diameters of around 80 nm and 120 nm when dispersed in incubation medium in the absence and the presence of 10% fetal calf serum, respectively. Acute exposure of neurons with IONPs for up to 6 h did neither alter the cell morphology nor compromise cell viability, although neurons accumulated large amounts of IONPs in a time- and concentration-dependent manner which caused delayed toxicity. For the first 30 min of incubation of neurons at 37 °C with IONPs the cellular iron content increased proportionally to the concentration of IONPs applied irrespective of the absence and the presence of serum. IONP-exposure in the absence of serum generated maximal cellular iron contents of around 3000 nmol iron/mg protein after 4 h of incubation, while the accumulation in the presence of 10% serum was slower and reached already within 1 h maximal values of around 450 nmol iron/mg protein. For both incubation conditions was the increase in cellular iron contents significantly lowered by reducing the incubation temperature to 4 °C. Application of inhibitors of endocytotic pathways did not affect neuronal IONP accumulation in the absence of serum, while inhibitors of clathrin-mediated endocytosis lowered significantly the IONP accumulation in the presence of serum. These data demonstrate that DMSA-coated IONPs are not acutely toxic to cultured neurons and that a protein corona around the particles strongly affects their interaction with neurons.

Comparison of Primary and Secondary Rat Astrocyte Cultures Regarding Glucose and Glutathione Metabolism and the Accumulation of Iron Oxide Nanoparticles

Astrocyte-rich primary cultures (APCs) are frequently used as a model system for the investigation of properties of brain astrocytes. However, as APCs contain a substantial number of microglial and oligodendroglial cells, biochemical parameters determined for such cultures may at least in part reflect also the presence of the contaminating cell types. To lower the potential contributions of microglial and oligodendroglial cells on properties of the astrocytes in APCs we prepared rat astrocyte-rich secondary cultures (ASCs) by subculturing of APCs and compared these ASCs with APCs regarding basal metabolic parameters, specific enzyme activities and the accumulation of iron oxide nanoparticles. Immunocytochemical characterization revealed that ASCs contained only minute amounts of microglial and oligodendroglial cells. ASCs and APCs did not significantly differ in their specific glucose consumption and lactate production rates, in their specific iron and glutathione contents, in their specific activities of various enzymes involved in glucose and glutathione metabolism nor in their accumulation of iron oxide nanoparticles. Thus, the absence or presence of some contaminating microglial and oligodendroglial cells appears not to substantially modulate the investigated metabolic parameters of astrocyte cultures.

Toxicity of dimercaptosuccinate-coated and un-functionalized magnetic iron oxide nanoparticles towards aquatic organisms

Magnetic iron oxide nanoparticles (IONP) have gained growing attention in recent years for their promising applications in medical treatment and environmental remediation. Among the IONP, dimercaptosuccinic acid coated-IONP (DMSA-IONP) have great potential because of their rapid uptake into cells and their potential to effectively adsorb heavy metals. The widespread use and potential release of IONP into the environment raises concern about their environmental impact. To date, little is known about the consequences of the exposure of aquatic organisms to such particles. In this context we investigated the colloidal stability of DMSA-IONP in different test media as well as their effects on green algae (Raphidocelis subcapitata), duckweed (Lemna minor) and water fleas (Daphnia magna). Moreover, a comparative analysis of stability and ecotoxicity data of DMSA-IONP with freshly prepared and aged uncoated IONP dispersions was performed, considering the importance of stability of particles in determining their toxicity. The green algae were the most sensitive organism with EC50 values (72 h) ranging between 0.86–2.27 μM Fe (i.e. 0.05 to 0.13 mg Fe L−1) for the three types of IONP. The observed flocculation and (co-)sedimentation of algae with IONP are assumed to reduce the access of cells to nutrients and light. Lemna was not affected by any of the IONP due to the low availability of IONP induced by fast aggregation of IONP in the medium. Minor toxic effects on Daphnia were found for uncoated IONP (EC50 between 374–1181 μM Fe, i.e. 21–66 mg Fe L−1) after 72 h. However, the ingestion and accumulation of coated and uncoated IONP in the gastrointestinal tract of daphnids was observed. Our evaluation of IONP has revealed a certain hazard potential to aquatic organisms. In this light it appears important to prevent the release of large amounts of IONP into the environment, which might limit their applicability.

Reporter Dyes Demonstrate Functional Expression of Multidrug Resistance Proteins in the Marine Flatworm Macrostomum lignano: The Sponge-Derived Dye Ageladine A Is Not a Substrate of These Transporters

The marine plathyhelminth Macrostomum lignano was recently isolated from Adriatic shore sediments where it experiences a wide variety of environmental challenges, ranging from hypoxia and reoxygenation, feeding on toxic algae, to exposure to anthropogenic contaminants. As multidrug resistance transporters constitute the first line of defense against toxins and toxicants we have studied the presence of such transporters in M. lignano in living animals by applying optical methods and pharmacological inhibitors that had been developed for mammalian cells. Application of the MDR1 inhibitor Verapamil or of the MRP1 inhibitors MK571 or Probenecid increased the intracellular fluorescence of the reporter dyes Fura-2 am, Calcein am, Fluo-3 am in the worms, but did not affect their staining with the dyes Rhodamine B, CMFDA or Ageladine A. The marine sponge alkaloid Ageladine A remained intracellularly trapped for several days in the worms, suggesting that it does not serve as substrate of multidrug resistance exporters. In addition, Ageladine A did not affect multidrug resistance-associated protein (MRP)-mediated dye export from M. lignano or the MRP1-mediated glutathione (GSH) export from cultured rat brain astrocytes. The data obtained demonstrate that life-imaging is a useful tool to address physiological drug export from intact marine transparent flatworms by using multiphoton scanning microscopy.

Uptake, metabolism and toxicity of iron oxide nanoparticles in cultured microglia, astrocytes and neurons

Iron oxide nanoparticles (IONPs) are frequently used for biomedical applications including magnetic resonance imaging, drug delivery or tumor treatment by hyperthermia. Since IONPs can reach the brain from periphery or are directly applied into the brain, the investigation of potential adverse effects of IONPs on properties and functions of the different types of brain cells is an important task. In order to directly compare different types of brain cells concerning the uptake and toxicity of IONPs, we synthesized fluorescent IONPs and characterized these BODIPY-labeled particles regarding their physicochemical properties. In the medium used for the cell studies, these particles had a hydrodynamic diameter of around 158 ± 28 nm and a negative surface charge with a zeta-potential of -10 ± 2 mV. Exposure of primary cultures of rat astrocytes, neurons and microglial cells revealed that among these cell types, microglial cells accumulated IONPs most rapidly. However, microglial cells were also most vulnerable towards acute IONP-induced stress while astrocytes and neurons were not acutely damaged by IONPs. In microglial cells, but not in astrocytes or neurons, IONPs were found to be localized in lysosomes and large amounts of reactive oxygen species (ROS) were observed after IONP-exposure. The IONP-induced toxicity in microglial cells was prevented by neutralizing lysosomes or by chelation of intracellular ferrous iron ions, suggesting that the toxic potential of IONPs in microglia involves rapid particle uptake, liberation of ferrous iron from the internalized IONPs in the acidic lysosomal compartment and iron-catalyzed ROS formation. These data suggest that also in brain IONPs may harm microglial cells and compromise microglial functions.