Nabeshi et al. Nanoscale Research Letters 2011, 6:93 http://www.nanoscalereslett.com/content/6/1/93 industry in the longer term by helping the researchers to protect both individuals and the environment from potentially damaging materials. Some recent articles have focused on the possible influence of surface charge in terms of the cellular uptake and/or cytotoxicity of nanoparticles [16-19]. Mayer et al. [19] reported the activation of the comple- ment system and increased hemolysis in blood samples after exposure to positively charged polystyrene nano- particles. Some recent studies suggest that cationic nanoparticles elicit a greater cytotoxicity compared with anionic nanoparticles [20-22]. Taken together, these stu- dies indicate that the surface property of nanoparticles is an important factor when developing safer forms of NMs. However, studies of cellular responses to NMs often give conflicting results. The aim of this study was to investigate the cytotoxicity caused by exposure of a murine macrophage cell line (RAW264.7) to silica nano- particles whose surface was either unmodified (nSP70) or modified with amine (nSP70-N) or carboxyl groups (nSP70-C). The intracellular localization of the different nanoparticles was also examined. Experimental procedures Silica particles Fluorescent (red-F or green-F)-labeled silica particles with surfaces that were either unmodified or modified with amine or carboxyl groups (Micromod Partikeltech- nologie GmbH, Rostock, Germany; designated nSP70, nSP70-N, and nSP70-C, respectively) were used in this study. The silica particles, which had a diameter of 70 nm, were prepared as a suspension (25 mg/ml) and sonicated for 5 min and then vortexed for 1 min imme- diately prior to conducting each experiment. Physicochemical examination of the nanosilica preparations Nanosilicas were diluted to 0.25 mg/ml with water, and the average particle size and zeta potential were mea- sured using a Zetasizer Nano-ZS (Malvern Instruments Ltd., Malvern, UK). The mean size and the size distribu- tion of silica particles were measured by dynamic light scattering. The zeta potential was measured by laser Doppler electrophoresis. Cell culture The mouse macrophage cell line, RAW 264.7, was obtained from the American Type Culture Collection. RAW 264.7 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% heat-inactivated FCS, 1% Antibiotic-Antimycotic Mix stock solution (GIBCO, CA, USA). All cultures were incubated at 37°C in a humidified atmosphere with 5% CO2. Page 2 of 6 3H-Thymidine incorporation assay The proliferation of nanosilica-treated RAW 264.7 cells and untreated cells was measured using a 3H-thymidine incorporation assay. 104 cells were cultured with varying concentrations of nanosilica diluted with medium for 18 h at 37°C, and 3H-thymidine (1 μCi/well) was then added into each well. After a further 6 h, cells were har- vested and lysed on glass fiber filter plates using a Cell harvester (Perkin-Elmer, Wellesley, MA, USA). The fil- ter plates were then dried and counted by standard liquid scintillation counting techniques in a TopCounter (Perkin-Elmer). Confocal scanning laser microscopy analysis of the macrophage cell line RAW 264.7 cells were cultured with nSP70, nSP70-N, and nSP70-C (100 μg/ml) for 3 h on chamber slides, then fixed at room temperature in 4% paraformaldehyde and washed three times in 0.1 M phosphate buffer (pH 7.4). Cells were then filled with mounting medium containing 4’,6-diamino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). A glass cover slip was then placed on the slide and fixed with glue. The mounted slides were examined under a confocal scan- ning laser microscope (Leica Microsystems, Mannheim, Germany). Results and discussion First, the authors assessed the mean particle size and surface charge of 70 nm silica particles in water whose surface was unmodified (nSP70) or chemically modified with amine (nSP70-N) or carboxyl groups (nSP70-C). The results are summarized in Table 1. Mean particle sizes of nSP70, nSP70-N, and nSP70-C as measured by dynamic light scattering method were 64.2 ± 0.6, 72.7 ± 1.3, and 76.2 ± 1.6 nm, respectively. These experimen- tally determined particle sizes were almost equal to the primary diameter sizes (70 nm). Surface charges (zeta potential) of nSP70-N and nSP70-C were, respectively, higher and lower compared to those of nSP70. Cytotoxicity of the three nanosilicas was tested by monitoring the incorporation of 3H-thymidine into RAW 264.7 cells. nSP70 showed the highest cytotoxicity (EC50 value = 121.5 μg/ml), while nSP70-N and nSP70- Table 1 Average particle size and zeta potential of unmodified and modified nanosilica nSP70 nSP70-N nSP70-C Modification substance - NH2 COOH Mean particle size in water (nm) 64.2 ± 0.6 72.7 ± 1.3 76.2 ± 1.6 Mean zeta potential (mV) -42.1 ± 0.6 -29.8 ± 0.5 -72.0 ± 1.9 Each value represents the mean ± SD.