5-(N-Ethyl-N-isopropyl)-Amiloride

Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells

Eva M. Luther a,b, Charlotte Petters a,b, Felix Bulcke a,b, Achim Kaltz a, Karsten Thiel c, Ulf Bickmeyer d, Ralf Dringen a,b,⇑
aCenter for Biomolecular Interactions Bremen, University of Bremen, P.O. Box 330440, D-28334 Bremen, Germany
bCenter for Environmental Research and Sustainable Technology, Leobener Strasse, D-28359 Bremen, Germany
cFraunhofer Institute for Manufacturing Technology and Advanced Materials, Wiener Strasse 12, D-28359 Bremen, Germany
dAlfred-Wegener-Institut für Polar und Meeresforschung, Am Handelshafen 12, D-27570 Bremerhaven, Germany

Article history:
Received 27 October 2012
Received in revised form 17 May 2013 Accepted 21 May 2013
Available online 31 May 2013
Keywords: Endocytosis Iron Lysosomes Microglia Nanoparticles

a b s t r a c t

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 BODIPYti-labelled IONPs (BP-IONPs). The aver- age hydrodynamic diameter and the f-potential of BP-IONPs in water were ti65 nm and ti49 mV, respec- tively. Both values increased after dispersion of the particles in serum containing incubation medium to ti 130 nm and ti8 mV. Exposure of cultured rat microglial cells with BP-IONPs caused a time-, concentra- tion- and temperature-dependent uptake of the particles, as demonstrated by strong increases in cellular iron contents and cellular fluorescence. Incubation for 3 h with 150 and 450 lM iron as BP-IONPs increased the cellular iron content from a low basal level of ti50 nmol iron mgti1 to 219 ± 52 and 481 ± 28 nmol iron (mg protein)ti1, respectively. These conditions did not affect cell viability, but expo- sure 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, tyrphostin 23 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

1.Introduction

Iron oxide nanoparticles (IONPs) are used for neurobiological applications, including cancer treatment by hypothermia, as con- trast agents for magnetic resonance imaging (MRI) as well as for targeted drug delivery and cell transfection [1,2]. Direct access of IONPs to brain tissue is achieved by injection into the affected brain area for treatment of brain tumours [3]. However, IONPs that are administered peripherally by oral application, intravenous injection or by inhalation have been reported to enter the brain by crossing the blood–brain barrier or via the olfactory system [1,2,4–6].Microglial cells are the immune competent cells of the brain. Depending on the situation, microglial cells can be beneficial or harmful to their neighbouring cells. In the healthy adult brain, so-called ‘‘resting’’ microglia survey their microenvironment for nutrients or debris, release neurotrophic factors and anti-inflam- matory cytokines, and promote synaptic plasticity [7,8]. However, upon activation by brain injury or infections, microglial cells mi- grate to the site of the impact and secrete inflammatory proteins and reactive oxygen species (ROS) that may damage neighbouring cells [7,9]. Microglial cells will encounter nanoparticles that have entered the brain, since these cells are known to literally scan their surroundings for debris and particles which are subsequently ta- ken up [7–9]. Indeed, exposure of animals with IONPs as contrast agents for MRI revealed that in the brain especially the microglial cells are strongly labelled [10–13].
Metal-containing nanoparticles (NPs), such as IONPs, titan diox- ide NPs, gold NPs, alumina NPs or quantum dots, have been re- ported to affect microglial functions in vivo and have been by microglial cells. A few studies have used microglial cell lines as model systems to gain information on the consequences of an exposure of microglial cells with IONPs [12,19–22]. However, it has to be considered that the advantageous feature of immortality of cell lines may be accompanied by properties and behaviours that differ from those of primary cells [8,23].
For previous studies on the accumulation of IONPs by cultured brain cells, we used non-fluorescent dimercaptosuccinate (DMSA)- coated IONPs [24,25]. However, visualization of the presence of such particles in cells is difficult and requires electron microscopy. This limitation can be bypassed in part by using fluorescent IONPs for uptake studies; this allows us to obtain information on the intracellular localization of the accumulated IONPs by fluorescence microscopy. As a tool for such studies, we have synthesized fluo- rescent BODIPYti -labelled IONPs (BP-IONPs) by coating IONPs with BP-labelled DMSA and have characterized these particles for their physicochemical properties and their colloidal stability. With the BP-IONPs generated, we were able to apply high concentrations of IONPs to cultured cells for detailed iron accumulation studies and could also investigate the cellular localization of the accumu- lated BP-IONPs by fluorescence microscopy.
Cells in secondary microglial cultures have been shown to accu- mulate fluorescent IONPs by analysis of their cellular fluorescence [13,26]. However, to our knowledge, no detailed quantitative anal- ysis of IONP uptake into microglial cells or identification of mech- anisms involved in particle uptake have been reported so far. Here we show by quantitative iron determination, cytochemical iron staining and fluorescence microscopy that cultured primary microglial cells efficiently accumulated BP-IONPs in a time-, con- centration- and temperature-dependent manner by endocytotic processes, which direct the accumulated particles into the lyso- somal compartment.

2.Materials and methods

2.1.Materials

Fetal calf serum (FCS), trypsin solution and penicillin/strepto- mycin solution were obtained from Biochrom (Berlin, Germany). Dulbecco’s modified Eagle’s medium (DMEM) was from Gibco (Karlsruhe, Germany) and 4-(2-hydroxyethyl)-1-piperazine eth- anesulfonic acid (HEPES) from Roth (Karlsruhe, Germany). Bovine serum albumin and nicotinamide adenine dinucleotide were from Applichem (Darmstadt, Germany). BODIPYti FL C1-IA [N-(4,4-di- fluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl) iodoacetamide] and lysotracker Red DND-99 were purchased from Invitrogen (Darmstadt, Germany). 5-(N-ethyl-N-isopropyl)amilo- ride (EIPA), tyrphostin 23, ferrozine, dimercaptosuccinic acid (DMSA), 40 ,6-diamidino-2-phenylindol hydrochloride (DAPI) and paraformaldehyde were purchased from Sigma–Aldrich (Stein- heim, Germany). Mouse anti-rat CD11b (Ox-42) antibody was pur- chased from Serotec (Düsseldorf, Germany) and the Cy3- conjugated anti-mouse immunoglobulin from Dianova (Hamburg, Germany). Other chemicals of the highest purity available were purchased from Fluka (Buchs, Switzerland) or Merck (Darmstadt, Germany). 96-well microtitre plates and 6-well cell culture plates were from Nunc (Wiesbaden, Germany) and 24-well cell culture plates from Sarstedt (Nümbrecht, Germany).

2.2.Synthesis and characterization of BP-IONPs

IONPs were synthesized by chemical co-precipitation of ferrous and ferric iron salts as described previously [27]. The nanoparticles were coated with DMSA or BODIPYti (BP)-labelled DMSA according to a modification [24] of a published method [28]. BP-DMSA was synthesized by thoroughly mixing 712.5 lM BODIPYti FL C1-IA and 4.75 mM DMSA at pH 10 in 10 mM NH3 or in 47.5 mM gly- cine/NaOH buffer. A 30 min incubation at room temperature (RT) led to the complete derivatization of BODIPYti FL C1-IA with thiol groups of DMSA (Fig. S.1). Electrospray ionization mass spectrom- etry revealed the expected signals at 470 and 759 m/z for DMSA la- belled with BP on one or both thiol groups, respectively (Fig. S.1). IONPs were added to the BP-DMSA reaction mixture to a final con- centration of 21.4 mM and the mixture was acidified to pH 3 by concentrated HNO3. After mixing for 30 min at RT, the particles were separated from the solution by magnetic force, resuspended in H2O and redispersed by increasing the pH value with NaOH to pH 9–10. Finally, the pH was lowered to 7.4 by adding HCl. This dispersion was diluted with water to a final iron concentration of 40 mM and stored at 4 tiC. Fourier transform infrared spectroscopy confirmed the presence of the coating material in the BP-IONPs generated (Fig. S.2). The concentrations of BP-IONPs used in the individual experiments are given here as concentrations of the iron present in the nanoparticle dispersion and do not represent the concentration of particles.
Samples for transmission electron microscopy (TEM) were pre- pared by dropping 5 ll of 1 mM BP-IONP dispersion in water onto carbon-coated copper grids and subsequent air drying at RT. Images were taken by a FEI Tecnai F20 S-TWIN (Hillsboro, Oregon, USA) operated at 200 kV and equipped with a GATAN GIF2001 SSC- CCD camera. Energy-dispersive X-ray analysis (EDX) was used for elemental analysis in the scanning mode of the microscope (STEM) with an EDAX r-TEM-EDX-detector with an energy resolution of 136 eV. The hydrodynamic diameters and the f-potentials of 1 mM BP-IONPs dispersed in different media were determined at 25 tiC by dynamic and electrophoretic light scattering in a Beckman Coulter (Krefeld, Germany) DelsaTM Nano C particle analyser at scattering angles of 165 and 15ti, respectively. The fluorescence spectra of diluted BP-IONP solutions (50 lM in water) were re- corded using a Cary Eclipse fluorimeter (Varian, Darmstadt, Germany).
The hydrodynamic diameter, f-potential and the fluorescence intensity of the dispersed BP-IONPs did not change during storage for at least up to 1 month, nor was release of any low molecular weight iron from the particles detectable during storage (data not shown).

2.3.Cell cultures

Primary microglial cultures were prepared from astroglia-rich primary cultures by tryptic removal of the astrocyte layer using a modification of a published method [29]. Astroglia-rich primary cultures were prepared from the whole brains of neonatal Wistar rats [30] and 300,000 viable cells were seeded per well of a 24-well plate with or without coverslips in 1 ml culture medium (90% DMEM, 10% FCS, 20 U mlti1 of penicillin G and 20 lg mlti1 of streptomycin sulfate) or 1.5 ti 106 cells per well of a six-well plate in 2.5 ml medium. The cultures were grown in a cell incubator (Sa- nyo, Osaka, Japan) that contained a humidified atmosphere of 10% CO2/90% air and the culture medium was renewed every sev- enth day. To obtain microglial cultures, confluent 14- to 23-day-old astroglia-rich cultures were incubated for 30 min with 0.5% (w/v) trypsin in serum-free DMEM. This treatment resulted in the detachment of an intact top layer of cells that contain virtually all the astrocytes and left a population of firmly attached microglial cells in the wells. The microglial cells were washed with 2 ml cul- ture medium and cultured in 1.5 ml glia-conditioned medium (GCM; 0.2 lm filtered glia-conditioned culture medium harvested after 1 day of incubation of astroglia-rich primary cultures) for additional 16–20 h before experiments were performed. The cul- tures obtained by this method are highly enriched in microglial cells, as more than 98% of the cells in these cultures are positive for the microglial marker protein CD11b.

2.4.Experimental incubations

Unless otherwise stated, microglial cultures on six-well dishes were incubated at 37 tiC with 1 ml of GCM containing BP-IONPs and other compounds in the concentrations indicated in the leg- ends of the figures and tables. To test for the temperature depen- dence of BP-IONP uptake, microglial cells were incubated at the given temperature with 1 ml GCM containing 20 mM HEPES (ad- justed to pH 7.4 by addition of 5 M NaOH). After the desired incu- bation period, the media were collected for measurement of extracellular lactate dehydrogenase (LDH) activity and the cells were washed once with 1 ml of ice-cold phosphate-buffered saline (PBS: 10 mM potassium phosphate buffer pH 7.4, containing 150 mM NaCl). Cells were either lysed in 1 ml 1% (w/v) Triton X- 100 in serum-free DMEM for analysis of the LDH activity or in 1 ml 1% (w/v) sulfosalicylic acid for determination of the glutathi- one content, fixed in 3.5% (w/v) paraformaldehyde in PBS for microscopy or stored dry at ti20 tiC until quantification of their iron and protein contents.

2.5.Determination of cell viability, protein content and glutathione content

The viability of microglial cultures was determined by quantifi- cation of cellular and extracellular activity of the cytosolic enzyme lactate dehydrogenase (LDH) or by investigating the membrane permeability for propidium iodide (PI). LDH activity was deter- mined as previously described [31] with the modification that 180 ll of lysates or media were used in the assay. For analysis of the membrane permeability the cells were incubated with PI as de- scribed previously [32]. To visualize all cell nuclei present, the cells were counterstained with the membrane-permeable Hoechst-dye H33342. An increase in the number of PI-positive cells or in the amount of extracellular LDH reflects a loss in cell viability. The pro- tein content of the cultures was determined according to the Lowry method [33] after solubilization of the cells in 700 ll of 50 mM NaOH using bovine serum albumin as a standard. The contents of total glutathione (GSx, amount of GSH plus two times the amount of glutathione disulfide (GSSG)) and GSSG in cell lysates were determined by a modified colorimetric Tietze assay [34]. Control experiments revealed that none of these colorimetric assays was affected by the presence of IONPs (data not shown).

2.6.Iron quantification and histochemical Perls’ staining for iron

The total iron content of cells, media or particle dispersions was determined by a colorimetric ferrozine method as previously de- scribed [24]. Iron was also cytochemically visualized by a modifica- tion of the histochemical Perls’ staining as previously described [27]. For the co-localization of cellular BP-IONP fluorescence with Perls’ staining, the fluorescence pictures were taken before the iron staining was completed by diaminobenzidine-nickel enhancement.

2.7.Activation of microglial NADPH oxidase

Microglial cells were incubated with 0.5 ml of 1 mM nitroblue tetrazolium chloride in GCM with or without 150 lM BP-IONPs for 3 h. To some wells 50 nM phorbol 12-myristate 13-acetate was added after 2 h of incubation as a positive control for the acti- vation of NADPH oxidase [35]. The formation of blue formazan crystals was monitored with an Eclipse TE-2000U fluorescence microscope (Nikon, Düsseldorf, Germany).

2.8.Immunocytochemical staining

Cells grown on coverslips in wells of 24-well dishes were washed once with 1 ml ice-cold PBS and fixed with 3.5% (w/v) paraformalde- hyde in PBS for 10 min at 4 tiC. Unless otherwise stated, the cells were washed thrice (5 min each) with PBS between the different steps of the staining procedure. After incubating the fixed cells with 0.1% (w/v) glycine in PBS for 5 min at RT, the membranes of the cells were permeabilized with 0.3% Triton X-100 in PBS for 10 min at RT. Incubation of the cells with mouse anti-CD11b (1:100 diluted in PBS) was carried out for 2 h at RT in a humidified atmosphere, fol- lowed by an incubation with the secondary Cy3-coupled goat anti- mouse antibody (1:200 diluted in PBS) for 30 min at RT. For visual- ization of the nuclei, the cells were treated with DAPI (1 mg mlti1 in PBS) for 5 min at RT. Prior to mounting the coverslips in Mowiol mounting media, an ethanol gradient of 70, 90 and 100% in 1 min intervals was applied. Fluorescence images were taken by using the Eclipse TE-2000U fluorescence microscope.
For co-localization of accumulated BP-IONPs with lysotracker, the cells were incubated with 150 lM iron as BP-IONPs for 3 h. 75 nM lysotracker was applied for the last hour of the incubation with BP-IONPs. Alternatively, after 3 h incubation with BP-IONPs and a further 90 min incubation in IONP-free GCM, the cells were incubated with lysotracker for 1 h. After the incubation with lyso- tracker, the cells were washed once with 1 ml of PBS and fixed with 3.5% (w/v) paraformaldehyde for 10 min. The images of cellular fluorescence for BP-IONPs (excitation: 488 nm, emission: 500– 520 nm) and lysotracker (excitation: 561 nm, emission: 590– 620 nm) were taken by a Leica SP5 confocal laser scanning micro- scope (Leica, Wetzlar, Germany).

2.9.Presentation of data

Unless otherwise stated, quantitative data are presented as means ± SD of values from at least three experiments that were performed on independently prepared cultures. Analysis of signif- icance of the differences between groups of data was performed by ANOVA followed by the Dunnetts’ post hoc test. The significance of difference between two sets of data was analysed by the t-test. p > 0.05 was considered as not significant.

3.Results

3.1.Characterization of BP-IONPs

BP-IONPs were synthesized as described in the methods section. TEM analysis revealed that the synthesized BP-IONPs displayed a spherical morphology with a particle diameter of 5 to 20 nm (Fig. 1A). EDX analysis confirmed the presence of iron and sulfur, demonstrating successful coating of IONPs with the sulfur-contain- ing BP-DMSA (Fig. 1B). Presence of the fluorescent dye BP in BP-ION- Ps was confirmed by fluorescence spectroscopy of the particles that showed maxima of 490 nm and 510 nm in the excitation and emis- sion spectra, respectively (Fig. 1C). The average hydrodynamic diam- eter, the polydispersity index and the f-potential of BP-IONPs dispersed in water were 65 ± 4 nm, 0.214 ± 0.024 and ti49 ± 2 mV, respectively (Fig. 1D; Table 1). These physicochemical parameters of BP-IONPs did not significantly differ (n = 3–5) from those previ- ously determined (59 ± 9 nm, ti77 ± 18 mV) for the respective non- fluorescent DMSA-coated IONPs [24].
For cell experiments, IONPs have to be dispersed in physiological media or buffers to maintain the cell viability. Since the composition of the medium can strongly affect the properties of engineered NPs [36], we investigated the effects of different media on the size and the f-potential of BP-IONPs. Dispersion of BP-IONPs in plain DMEM culture medium caused rapid precipitation of the particles.

Fig. 1. Characterization of BP-IONPs. (A) Transmission electron microscopic picture of BP-IONPs. (B) Energy dispersive X-ray spectrum of BP-IONPs. (C) Emission (excitation at 490 nm) and excitation (emission at 510 nm) fluorescence spectra of a 50 lM dispersion of BP-IONPs in water. (D) Intensity distribution of the hydrodynamic diameter of BP-IONPs (1 mM) dispersed in water or GCM as determined by dynamic light scattering.

Table 1
Hydrodynamic diameter and f-potential of BP-IONPs in different media.
The data represent mean values ± SD of n independently performed experiments.
This precipitation was not observed for DMEM containing 10% FCS or for GCM that also contained 10% FCS. For these conditions, the diameter of the particles was doubled (to ti130 nm) compared to that determined for the water dispersion (65 nm), while the polydispersity index became slightly increased to 0.275 ± 0.018 and the f-potential became more positive, to
titi9 mV (Table 1). Non-fluorescent DMSA-coated IONPs that had been dispersed in GCM had an average hydrodynamic diameter and a f-potential (n = 3) of 125 ± 20 nm and ti11 ± 1 mV, respec- tively, and thus did not differ significantly in these parameters to BP-IONPs that had been dispersed in GCM (Table 1).

3.2.Characterization of microglial cell cultures

The primary microglial cultures prepared by the trypsinization method [29] contained cells with the typical amoeboid or bipolar morphologies (Fig. 2A) that have previously been described for cul- tured microglia [8,9,29]. Immunocytochemical staining with the antibody Ox-42 for the microglial marker protein CD11b [8,26]
demonstrated that almost all cells in the cultures were positive for this microglial marker (Fig. 2B).

3.3.Viability of microglial cells after exposure to BP-IONPs

To test for the consequences of an exposure of cultured microg- lial cells to BP-IONPs, the cells were incubated for up to 6 h in GCM with BP-IONPs. While the incubation of microglial cells without BP-IONPs for 6 h (Fig. 3A and B) or with up to 150 lM BP-IONPs for 3 h (Fig. 3D and E) did at best marginally affect cell viability (Fig. 3A and D) and the cellular protein content (Fig. 3B and E), the incubation of microglial cells for more than 3 h with 450 or 1500 lM iron as BP-IONPs severely compromised the cell viability as shown by the significant loss in cellular LDH activity and by the accompanying increase in extracellular LDH activity (Fig. 3A and D). The compromised viability of microglial cells that had been treated with higher concentrations of BP-IONPs (Fig. 3D) was confirmed by PI staining (Fig. 4). After a 3 h exposure to 1500 lM iron as BP-IONPs the membranes of the majority of cells were per- meable for the dye (Fig. 4K).
Fig. 2. Characterization of primary microglial cultures. (A) Light microscopical image of a microglia-rich culture. (B) Immunocytochemical staining of the cultures for the microglial marker protein CD11b. Nuclei (arrows) were counter-stained with DA
Fig. 3. Consequences of an exposure of microglial cultures to BP-IONPs. The cells were incubated without (0 lM) or with 150 or 450 lM BP-IONPs for up to 6 h (A–C) or for 3 h with the indicated concentrations of BP-IONPs (D-F) and the cellular and extracellular LDH activities (A, D), the protein content (B, E) as well as the specific iron content of the cells (C, F) were determined. Indicated is the significance of differences between the values obtained for BP-IONP-treated cells compared with controls (absence of BP- IONPs) (⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001). Accumulated IONPs have been reported to induce oxidative stress in cultured cells [37,38]. To test whether cultured microg- lial cells may suffer from oxidative stress during exposure to BP-IONPs, we tested for potential alterations in the cellular GSH redox state. However, microglial cells that had been exposed for 3 h to 150 or 450 lM iron as BP-IONPs had almost identical cel- lular GSx values as control cells and did not show any increase in cellular GSSG values (Table 2) that would indicate a severe oxida- tive stress. To investigate whether the coating material BP-DMSA of IONPs may contribute to the toxicity observed for high concentrations of BP-IONPs, microglial cells were incubated for 3 h without or with 20 lM BP-DMSA. This represents the concentration of coating material used for coating of an equivalent of 1500 lM IONPs. Treatment of microglial cells for 3 h with 20 lM BP-DMSA did not increase (n = 2) the extracellular LDH activity (5 ± 1% of total LDH activity) compared to the values determined for control cells (absence of BP-DMSA; 4 ± 1% of total LDH activity). 3.4.Accumulation of iron from BP-IONPs by cultured microglial cells Exposure of microglial cells to BP-IONPs caused a time- and concentration-dependent increase in the cellular iron content (Fig. 3C and F, Fig. 5), while the iron content of microglial cells incubated without BP-IONPs was not altered (Fig. 3C and F). After a 3 h incubation with 150 and 450 lM iron supplied as BP-IONPs, the cellular iron content was increased four- and tenfold to 219 ± 52 and 481 ± 28 nmol iron (mg protein)ti1, respectively, compared to the initial iron content of 49 ± 65 nmol iron (mg pro- tein)ti1 (Fig. 3C and F). During the 3 h incubation of microglial cells with 150 lM iron as BP-IONPs, the cells accumulated ti25% of the applied iron. Determination of physicochemical parameters of the remaining 75% of BP-IONPs that had not been taken up into the cells during the 3 h incubation (average hydrodynamic diame- ter of 113 ± 4 nm and f-potential of ti12 ± 3 mV) revealed that the colloidal stability of the particles was not altered during their incu- bation with microglial cells compared to the values of the particles dispersed in GCM that had no contact to the cells (Table 1). Visualization of the cellular iron content by cytochemical staining for iron by the Perls’ method confirmed for BP-IONPs the concentration-dependent increase in cell-associated iron (Fig. 5). Fig. 4. Effects of BP-IONPs on the membrane integrity of microglial cells. The cells were incubated without (A–C) or with 150 lM (D-F), 450 lM (G–I) or 1500 lM (J–L) iron as BP-IONPs for 3 h. Shown are the phase contrast images of the cells (A, D, G, J), the PI staining, which indicates nuclei of cells with permeabilized membranes (B,E,H,K) and the Hoechst 33342 (H33342) staining which identifies the nuclei of all cells present (C, F, I, L). The scale bar in L represents 100 lm and applies to all panels. Table 2 Effects of BP-IONPs on the glutathione content of cultured microglial cells. Cultured microglial cells were incubated for 3 h without (0 lM) or with 150 or 450 lM of iron as BP-IONPs and the specific contents of total glutathione (GSx) and GSSG as well as the protein content were measured. The data represent mean values ± SD of 3 experiments performed on independently prepared cultures.hardly contain Perls’-detectable iron (Fig. 5A), the dark staining of precipitates formed in BP-IONP exposed cells became more intense with increasing concentration of applied BP-IONPs (Fig. 5C, E and G). For all concentrations of BP-IONPs applied, the iron visualized by the Perls’ method was almost perfectly co-localized with the BP-fluorescence of the cells (Fig. 5). While microglial cells incu- bated without BP-IONPs did hardly show any fluorescence (Fig. 5B), the fluorescence intensity of the cells increased with the concentration of BP-IONPs applied (Fig. 5D, F and H). After incubation of the cultures with 450 lM BP-IONPs, all cells in the microglial cultures were Perls’ positive for iron (Fig. 5G) and showed a strong BP fluorescence (Fig. 5H). To test for a potential influence of the presence of the fluores- cent dye BP in the DMSA coat of IONPs, iron accumulation by microglial cells was compared for IONPs that had been coated with BP-DMSA or DMSA. The specific cellular iron content determined after a 3 h incubation of microglial cells with 150 lM BP-IONPs (305 ± 68 nmol mgti 1) was almost identical (n = 3, p > 0.05) to that found for cells which had been exposed to 150 lM DMSA-coated IONPs (305 ± 40 nmol mgti 1), demonstrating that the absence or presence of BP in the IONP coat does not affect the microglial accu- mulation of the IONPs.
To test for the influence of the incubation temperature on the accumulation of BP-IONPs by microglial cells, the cells were incu- bated with BP-IONPs in GCM that was pH stabilized at 4 tiC or 37 tiC by the addition of HEPES. Incubation of microglial cells in HEPES- stabilized GCM at 4 tiC neither altered the cellular protein content (Fig. 6B) nor compromised cell viability (Fig. 6C), but almost com- pletely prevented the accumulation of BP-IONPs by microglial cells (Fig. 6A). Treatment of microglial cells with the respective 37 tiC condition led to a substantial accumulation of BP-IONPs but the cell viability was also compromised as indicated by the increased extracellular LDH activity (Fig. 6C) compared to an incubation in HEPES-free GCM. This temperature dependence of BP-IONP accu- mulation in microglial cells was confirmed by Perls’ staining and fluorescence microscopy, which revealed that hardly any cell in microglial cultures that had been exposed to BP-IONPs at 4 tiC was Perls’ positive or showed BP fluorescence (data not shown).

3.5.Endocytosis and intracellular localization of BP-IONPs

Microglial cells contain a large number of lysosomal vesicles that can be stained with lysotracker (Fig. 7B). In the fluorescence channel used to detect BP-fluorescence, these cells showed only a weak autofluorescence (Fig. 7A) which was co-localized with the lysotracker-stained lysosomes (Fig. 7B). After exposure of the cells to 150 lM BP-IONPs for 3 h, microglial cells revealed a strong punctuated fluorescence staining (Fig. 7E), suggesting that most of the cellular BP-IONP fluorescence was associated with vesicular structures. Lysotracker co-staining revealed that only a part of these BP-IONP positive structures were lysosomes (Fig. 7F). How- ever, after a further 90 min incubation period of BP-IONP treated microglial cells in NP-free GCM the number of large BP-IONP posi- tive vesicles had increased (Fig. 7I and M) and the majority of these
vesicles were now positively stained with lysotracker (Fig. 7J, K, N and O).
Inhibitors of endocytotic pathways were used to investigate which pathways may be involved in the observed accumulation of BP-IONPs by microglial cells. The cellular iron content of microg- lial cells that had been exposed for 3 h to 150 lM iron as BP-IONPs (305 ± 68 nmol mgti1) was not significantly lowered (n = 3; p > 0.05) by the presence of the phagocytosis inhibitor cytochala- sin D [39] in a concentration of 500 nM (270 ± 56 nmol mgti1). In contrast, EIPA, a known inhibitor of macropinocytosis [40,41] as well as tyrphostin 23 and chlorpromazin, inhibitors of clathrin- dependent uptake [40–42], lowered the iron accumulation in BP- IONP treated microglial cells by ti30% compared to control condi- tions (Table 3), while a combination of these three inhibitors re- duced the cellular iron contents by almost 60% (Table 3). The presence of these inhibitors did not lower cell viability, as demon- strated by the absence of any significant increase in the extracellu- lar LDH activity (Table 3).

4.Discussion

4.1.Characterization of BP-IONPs

Fluorescent BP-IONPs were successfully synthesized and coated with BP-labelled DMSA as previously shown for unlabelled DMSA [24,43,44]. DMSA has been reported to form a cage-like structure around the IONPs by binding the carboxyl groups of DMSA to the nanoparticle surface and by the formation of disulfide bridges be- tween adsorbed DMSA molecules [28,45]. Complete derivatization of the BP applied labelled only 7.5% of the thiol groups of the avail- able DMSA, thereby leaving sufficient DMSA thiol groups for the formation of a disulfide-linked cage around the core of the IONPs. The characterization of BP-IONPs by TEM and dynamic light scat- tering revealed the presence of individual particles of ti10 nm in diameter which had formed small aggregates in dispersion, as pre- viously also reported for DMSA-coated IONPs [24]. Fluorescence spectroscopy confirmed the presence of the fluorescent dye BP in the particles, as BP-IONPs showed the fluorescence maxima around 490 nm and 510 nm in the excitation and emission spectra, respec- tively, that were also recorded for BP by us (data not shown) and others [46,47].
BP-IONPs were not stably dispersed in plain DMEM culture medium. The reason for this observation may be the high ionic strength of this medium and/or the presence of phosphate, which caused precipitation of DMSA-coated IONPs [48]. However, the presence of serum prevented the precipitation of BP-IONPs, most likely by forming a protein corona around the particles as previ- ously described for AgNPs [49,50] and non-fluorescent DMSA- coated IONPs [48,51,52]. This hypothesis is supported by the in- crease in hydrodynamic diameter as well as by the positivation of the f-potential of BP-IONPs dispersed in serum-containing med- ia. The presence of the fluorescent dye BP in the coat of BP-IONPs did not alter basal physicochemical parameters of the particles nor their colloidal stability in GCM compared to non-fluorescent

Fig. 5. Perls’ iron staining and BP-fluorescence of cultured microglial cells after exposure to BP-IONPs. The cells were incubated without (control, A, B) or with 45 lM (C, D), 150 lM (E, F) or 450 lM (G, H) of iron as BP-IONPs. Shown are overlays of the phase contrast images of the cells with the transmission light images of the Perls’ staining for cellular iron (A, C, E, G) and the corresponding BP fluorescence images (B, D, F, H) The size bar in panel H represents 50 lm and applies to all panels.

DMSA-coated IONPs, suggesting that BP-IONPs can be used as tool to reliably monitor by fluorescence microscopy the accumulation and cellular fate of DMSA-coated IONPs.

4.2.Microglial cell cultures
Highly purified microglial cultures were generated by tryptic removal of the astrocyte layer from astroglia-rich primary cultures.
Cell morphology and immunocytochemical staining confirmed lit- erature data [8,29] demonstrating that the cultures obtained were highly enriched for microglial cells. Although microglial cells in culture are described to be in an activated mode due to the artifi- cial environment [7], the microglial cultures used in our study could be further activated by incubation with a phorbol ester (data not shown) which induces superoxide generation by NADPH oxi- dase [35,53].
Fig. 6. Temperature-dependent uptake of iron from BP-IONPs by cultured microg- lial cells. The cells were incubated for 3 h without (control) or with 150 lM of iron as BP-IONPs in GCM at 37 tiC or in GCM-HEPES at 37 tiC or 4 tiC. The specific iron content (A), the protein content (B) and the extracellular LDH activity (C) were measured. Indicated is the significance of differences compared to the values obtained for cells that had been incubated at 37 ti C in GCM-HEPES (⁄⁄p < 0.01).observed for microglial cultures after exposure to BP-IONPs (data not shown), which confirms literature data that cultured microglial cells are not activated by an exposure to IONPs [26]. 4.3.Accumulation of BP-IONPs by viable microglial cells Exposure of microglial cells to moderate concentrations of BP- IONPs (up to 450 lM) for up to 3 h did not compromise cell viabil- ity, whereas higher concentrations of IONPs or elongated incuba- tion periods led to a severe loss in cell viability. This is consistent with literature data described for IONP-treated secondary microg- lial cultures [13,26]. Quantification of cellular iron contents of BP- IONP treated microglial cells revealed a concentration dependence of BP-IONP accumulation, which was confirmed by fluorescence microscopy and cytochemical Perls’ staining for iron. The strict co-localization of fluorescence and iron deposits suggests that un- der the conditions used most of the accumulated particles re- mained intact after uptake. Cultured viable microglial cells increased their cellular iron con- tent after a 3 h incubation with 450 lM BP-IONPs ten-fold. This cellular amount of accumulated IONPs of ti500 nmol (mg pro- tein)ti1, which represented less than 10% of the iron applied as BP-IONPs, appears to be the maximal amount of accumulated ION- Ps that can be tolerated by viable microglial cells, since higher con- centrations of IONPs or longer incubations that led to higher specific cellular iron contents compromised the cell viability. This observation is consistent with the view that microglial cells take up particles until they die [13]. The loss in cell viability upon expo- sure to high concentrations of IONPs is likely to be a consequence of a rapid liberation of iron ions from the accumulated particles, as low molecular weight iron has been described to be toxic for microglial cells [54,55] and to cause detachment of cells [56]. This hypothesis is consistent with the observation that cellular BP-IONP fluorescence in microglial cells was co-localized with lysosomes where the low pH has been described to facilitate IONP-degrada- tion by liberation of iron ions [57]. A contribution of the coating material BP-DMSA in the observed toxicity of higher concentra- tions of BP-IONPs appears to be unlikely, as the coating material without the iron oxide core did not compromise the viability of microglial cells. To test to what extent the determined cellular iron of IONP- treated microglial cells represented internalized iron or IONPs that were bound extracellularly to the cell membrane, the cells were exposed to BP-IONPs at 4 tiC, since this low temperature is known to slow down transport processes across the membrane and the internalization of IONPs [24,58,59]. After incubation at 4 tiC, only 20% of the cellular iron contents of the respective 37 tiC incubation were observed, suggesting that only ti20% of the cellular iron determined for BP-IONP treated microglial cells can be considered as externally attached. This is consistent with data obtained for the uptake of other types of fluorescent IONPs by microglial cells [26]. In contrast, membrane-attached IONPs represented ti50% of the to- tal cellular iron determined for IONP-treated astrocytes [24]. Thus, cultured microglial cells appear to be more efficient to internalize membrane-bound IONPs or are less efficient to bind IONPs than cultured astrocytes, although the specific capacity of cultured astrocytes to accumulate IONPs is much higher [24,58] than that of cultured microglial cells. 4.4.Mechanisms of BP-IONP uptake into microglial cells The uptake of BP-IONPs into vesicles and the subsequent co- localization of BP-IONPs in microglial cells with lysosomes suggest that endocytotic processes are involved in the accumulation of BP- IONPs into microglial cells. Although microglial cells have the capacity for phagocytosis [8], this process appears not to contrib- ute substantially to the observed BP-IONP accumulation by cul- tured microglial cells. At least the phagocytosis inhibitor cytochalasin D, which is described to inhibit a-synuclein-aggre- gate uptake in microglia [39], did not affect the BP-IONP accumu- lation by microglial cells. Since the aggregates of BP-IONPs formed in GCM had an average size of 140 nm, they may be too small to be taken up by phagocytosis, since this process has been discussed to be predominantly involved in uptake of particles larger than 200 nm [60], while smaller particles are taken up by endocytotic pathways [61]. Indeed, tyrphostin 23, chlorpromazin and EIPA, inhibitors of macropinocytosis and clathrin-dependent endocyto- sis [42,62,63] Fig. 7. Confocal co-localization of BP-IONPs and lysosomes in microglial cells. Microglial cells were incubated with lysotracker (LT) for 1 h either without prior incubation with BP-IONPs (control, A–D), during the last hour of a 3 h incubation with 150 lM BP-IONPs (E–H) or 90 min (I–P) after a 3 h loading of the cells with 150 lM BP-IONPs. The scale bars in L (applying to panels A–L) and P (applying to panels M–P) represent 10 lm. Table 3 Effects of endocytosis inhibitors on the cell viability and the accumulation of BP-IONPs by cultured microglial cells. Inhibitor/treatment Concentration (lM) Cellular LDH activity Extracellular LDH activity Protein content Specific iron content n (% of total) (% of total) (lg per well) (nmol mgti 1) (% of control) Control (1ti DMSO) 90 ± 11 10.4 ± 10.7 55 ± 12 257 ± 33 100 ± 0 3 EIPA 25 98 ± 1 1.5 ± 1.2 66 ± 22 186 ± 16⁄⁄ 73 ± 10⁄ 3 Tyrphostin 23 100 100 ± 1 0.1 ± 0.1 63 ± 18 191 ± 25⁄⁄ 74 ± 4⁄ 3 Chlorpromazine 10 99 ± 2 1.3 ± 1.5 70 ± 13 189 ± 9⁄⁄ 74 ± 7⁄ 3 Control (3ti DMSO) 98 ± 2 2.5 ± 2.3 76 ± 23 220 ± 83 100 ± 0 5 EIPA + 25 + Tyrphostin 23 + 100 + Chlorpromazin 10 98 ± 3 2.0 ± 2.1 67 ± 20⁄ 87 ± 26⁄⁄⁄ 42 ± 15⁄⁄⁄ 5 The cells were incubated for 3 h with 150 lM iron applied as BP-IONPs in the absence or the presence of the indicated endocytosis inhibitors. Since all inhibitors were applied from concentrated stock solutions in dimethyl sulfoxide (DMSO), the effect of the respective DMSO control was also investigated. The data represent mean values ± SD of n experiments performed on independently prepared cultures. Indicated are the significances between values obtained for cells treated with the indicated inhibitor(s) and the respective control (⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001). Endocytotic processes have also been de- scribed to mediate the uptake of IONPs into macrophages [64,65]. A contribution of phagocytosis in the uptake of at least carboxy- dextran-coated IONPs in peripheral macrophages was excluded [64], while the uptake of DMSA-coated IONPs into cells of the RAW cell line appears to involve multiple endocytotic pathways that may include also phagocytosis [65]. However, it should be stressed here that the specificity of inhibitors that are commonly used to identify endocytotic pathways is still under debate [40,41]. 4.5.Conclusions In summary, fluorescent BP-IONPs were synthesized and char- acterized as tools to investigate IONP uptake into cultured microg- lial cells. These cells efficiently accumulated BP-IONPs, as demonstrated by quantification of cellular iron contents as well as the co-localization of cellular iron and cellular fluorescence. The localization of BP-IONPs in lysosomes as well as the inhibition of BP-IONP uptake by endocytosis inhibitors demonstrates that the BP-IONPs are taken up into microglial cells by endocytosis and en- ter the lysosomal pathway. Although the low pH of the lysosomes may liberate some iron from the accumulated BP-IONPs, the cells remained viable after exposure to moderate concentrations of the particles and did not show any indications for enhanced activation or oxidative stress. Thus, microglial cells may act as a first defence line in brain that fast and efficiently takes up IONPs, thereby help- ing to protect the brain against damage by IONPs and IONP-derived iron. Further studies are now required to investigate whether BP- IONPs will also be a useful tool to study IONP uptake in brain cells in vivo. Acknowledgements E. M. Luther thanks the Hans-Böckler-Stiftung for her PhD fel- lowship. She is a member of the PhD graduate school nanoToxCom at the University of Bremen. The authors would like to thank Dr Thomas Dülks and Dorit Kemken (University of Bremen) for the mass spectrometric measurements and for their help with the analysis of the spectra and Professor Petra Swiderek (University of Bremen) for giving us access to her IR spectrometer. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 2, 5 and 7 are dif-ficult to interpret in black and white. 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