Analytical Chemistry behavior, thus tests on these substances must be done on an NM by NM basis. NM are deﬁned as substances with at least one dimension smaller than 100 nm, with diﬀerent physiochemical properties compared to their micrometer sized counterparts due to their high surface area. In some cases the small dimensions make NM more chemically reactive, with particle size inversely propor- tional to bioactivity and toxicity.15 19 High surface area can also change the strength and electrical conductivity of the material, while the quantum eﬀects associated with NM result in unique optical, electrical, and magnetic behavior. For example, when smaller than 20 30 nm, iron oxide nanoparticles (NP) become superparamagnetic; a property that makes this particular material very useful in a number of biomedical applications including magnetic drug targeting, as a contrast agent to enhance MRI imaging and in magnetic tumor ablation through hyperthermia.20 Given the potential clinical applications of ultraﬁne superpar- amagnetic iron oxide nanoparticles (USPION), evaluation of their safety is critical. Several studies already exist in the literature suggesting that iron oxide nanoparticles are toxic to cells and induce oxidative stress. For example, in 2003 Berry et al. showed that human dermal ﬁbroblast cells treated with either dextran coated- or uncoated-magnetite NP exhibit cell death and reduced proliferation.21 Another study reports that exposing iron oxide NP to human microvascular endothelial cells induces reactive oxygen species (ROS) production, that leads to the remodelling of microtubules and subsequently to increased cell permeability.22 In this study we investigated whether common assays used for the measurement of oxidative stress, cell viability, and cell growth are compatible with dextran coated ultraﬁne superparamagnetic iron oxide NP (dUSPION), to measure these parameters. We examined the interactions of dUSPION with cell viability assays: 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium (MTS), calcein, CyQUANT, and ethidium homodimer (EthD-1) in a cell free system. We also performed similar studies for oxidative stress assays: 20,70- dichloroﬂuorescein-diacetate (DCFH-DA) and 30-(p-aminophenyl) ﬂuorescein (APF). Furthermore, using the antioxidant N-acetyl cysteine (NAC), we examined the potential of dUSPION to initiate ROS production in a cell-free system, and using tert-butyl hydroperoxide (TBHP) as a source of ROS, we examined the potential antioxidant properties of magnetite. ’ MATERIALS AND METHODS Materials. dUSPION were purchased from Liquids research, Bangor, UK. RPMI 1640, horse serum, and Hanks balanced salt solution (with NaHCO3, without phenol red, calcium chloride, and magnesium sulfate) were purchased from Gibco, UK. Sodium hydroxide, glucose, sodium phosphate monobasic, and sodium phosphate dibasic were purchased from Fisher Scientific, UK. N-Acetyl-L-cysteine (NAC) and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich, UK. Tissue culture black microplates (96 well) were purchased from Greiner Bio-one, UK, and clear 96-well tissue culture microplates were purchased from Nunc, UK. CellTiter 96 Aqueous One Solution reagent was from Promega UK, Southampton, UK. CyQUANT probe, live/Dead Viability/cytotoxicity Kit, DCFH-DA, and APF were purchased from Invitrogen molecular probes, Paisley, UK. Methods. Preparation of dUSPION. dUSPION were supplied in suspension in water at a concentration of 10 mg/mL. dUSPION ARTICLE was diluted to the appropriate concentrations in distilled water and vortexed for 10 s immediately before use. Characterization of dUSPION. Dynamc Light Scattering ( DLS). The hydrodynamic particle size of dUSPION samples were obtained by DLS. The measurements were performed using a Malvern 4700 spectrometer (Malvern instruments Ltd., UK) either in RPMI with 1% horse serum or in Hepes-buffered (20 mM) Hanks balanced salt solution with glucose (5 mM) (pH 7.4). Data is presented as the average values of 15 readings. X-ray Photoelectron Spectroscopy (XPS). The samples were dried on an Indium substrate and examined with a PHI Quantera SXM(TM) (Ulvac-phi, Inc., Japan). All data points were ac- quired using a beam spot size of 200 um, 40 W, and 15 kV, under a pressure of 5 10 9 Torr. The electron source was Al mono- chromatic with a tilt angle of 45 , operating at 26 eV. Maghemite was acquired from 700 to 720 eV, using 70 sweeps with a band- pass energy of 26 eV. Oxygen was acquired from 525 to 537 eV, using 35 sweeps with a bandpass energy of 26 eV. Carbon was acquired from 278 to 293 eV, using 25 sweeps and a bandpass energy of 26 eV. Survey scans were completed from 0 to 1100 eV using 3 sweeps with a bandpass energy of 140 eV. Each sample was acquired on its own to prevent the possible contamination from previous samples. Zeta Potential. The z-potential values of the dUSPION were determined by Zetasizer 2000 (Malvern instruments Ltd., UK). The nanoparticles were prepared in water, and the z-potential values are presented as the average readings of 10 experiments. Transmisson Electron Microscopy (TEM). dUSPION sam- ples for TEM were prepared by dispersion in methanol, then drop-casting on holey carbon TEM support films (Cu-grids) and air-dried. TEM was performed using a Philips/FEI CM200 field emission gun TEM fitted with an Oxford Instruments ultrathin window EDX detector and ISIS software plus a Gatan Imaging Filter (GIF200) with Digitialmicrograph software. The micro- scope was operated at 197 keV. Viability and Oxidative Stress Assays. The first step for all assays was loading of dUSPION concentration range onto 96-well plates. After addition of the probes specified below, the fluores- cence or absorbance was measured on a POLARStar Omega plate reader (BMG Labtech, Aylesbury, UK). For all assays each dose of dUSPION was performed in triplicate within the plate, and each plate was performed in triplicate on three different days, thus accounting for both intra- and interplate variability, respectively. MTS Assay. The MTS assay it is based on the reduction of the tetrazolium compound MTS and an electron coupling reagent (phenazine ethosulfate; PES) into a soluble formazan product. This conversion takes place only in the presence of metabolically active cells, utilizing the mitochondrial dehydrogenase enzyme. The formazan product can be measured by absorbance at 490 nm, which is directly proportional to the number of live cells in culture and can thus be used for determining the number of viable cells in proliferation or cytoxicity assays. A 20 μl portion of CellTiter 96 Aqueous One Solution reagent was added to each well of a 96-well plate already loaded with 100 μl of dUSPION at different concentrations; then plates were incubated in a humidified incubator at 37 C for 1 h, and the absorbance was measured at 490 nm. CyQUANT Assay. A CyQUANT probe was prepared per the manufacturer’s instructions for use, 200 μl of this was added to the wells of a 96-well plate already loaded with dUSPION, and fluorescence was measured after 5 min (fluorescence excitation and emission at 480 and 520 nm, respectively). 3779 dx.doi.org/10.1021/ac200103x |Anal. Chem. 2011, 83, 3778–3785