Nanoscale Res Lett (2009) 4:70–77 oxide nanoparticles could be internalized by cells and induce a dramatic decrease in the metabolic activity and proliferation of human cells (MSTO-211H) [16–20]. A quantiﬁable model cell system shows that intracellular delivery of even moderate levels of iron oxide (Fe2O3) nanoparticles may adversely affect cell function. More speciﬁcally, the cytotoxicity studies show that exposure to increasing concentrations of anionic MNPs, from 0.15 to 15 mM of iron, results in a dose-dependent diminishing viability and capacity of PC12 cells to extend neurites in response to nerve growth factor [21]. Recently, the cytotoxicity assessment about iron oxide nanoparticles has been focused on by more and more researchers. Nevertheless, as mentioned by Auffan et al. [16], toxicological data are difﬁcult to compare since the parameters controlled in each of these studies may differ. These parameters involve size distribution, surface prop- erties, magnetic properties, stability in biological media, etc. In this present study, the aim is to elucidate the effect of different iron oxide magnetic nanoparticles (c-Fe2O3, MNPs) on Sprague-Dawley rat smooth muscle cell (SMC) in vitro. In particularly, MNPs were coated by meso-2, 3-dimercaptosuccinic acid (DMSA), 3-amino-propyltri- ethoxysilane (APTS), and L-glutamic acid (GLU), respectively, but possess the same size distribution and magnetic properties and stability, which can ensure the consistence and comparability of investigation results. Experimental Section Reagents Magnetic nanoparticles were synthesized, and stored in the dark at 4 C. Dulbecco’s modiﬁed Eagle’s medium (DMEM) was purchased from GIBCO Company. Penicillin and streptomycin were purchased SIGMA Company. TNF- a ELISA Kit was purchased from BOSTER Company. All of other chemicals were of reagent grade and were used as received without further puriﬁcation. Double distilled water was used for all the experiments. Preparation of the Coated MNPs Fe3O4 nanoparticles were synthesized by chemical copre- cipitation of Molday [22]. Typically, a solution of mixture of FeCl3 and FeSO4 (molar ratio 2:1) was prepared under N2 protecting, followed by the slow addition of enough ammonia aqueous solution with vigorous stirring for 30 min. The black Fe3O4 precipitates were obtained and washed immediately with distilled water for ﬁve times by magnetic separation. The ﬁnal precipitates were dispersed in distilled water with concentration of 0.128 M and pH 71 3.0, and oxidized into more stable MNPs (c-Fe2O3) by air at the temperature of 90 C. According to the process described in the literature [23, 24], MNPs were coated with DMSA and GLU. Finally stable aqueous sol DMSA-MNPs (DMSA-Fe2O3), and GLU-MNPs (GLU-Fe2O3) were obtained. Similarly, APTS-MNPs (APTS-Fe2O3) were prepared according to literature [25]. The part of above samples was dried into powder at room temperature under vacuum. Characterization The particle size and morphology of the coated MNPs was determined by transmission electronic microscopy (TEM, JEOL, JEM-200EX). Powder X-ray diffraction (XRD, Rigaku, D/Max-RA, k = 1.5405 9 10-10 m, CuK) was used to determine the crystal structure of MNPs. Surface charge measurements were performed with a zeta potential analyzer (BECKMAN, Delsa 440SX). The magnetic measurements were carried out with a vibrating sample magnetometer (VSM, Lakeshore 7407). Fourier transform infrared (FTIR) spectroscopy measurements were per- formed on a Bruker Fourier transform spectrometer model VECTOR22 using KBr pressed discs. Cell Culture Sprague-Dawley (SD) rat aortic SMCs were grown from explants of normal SD rat aorta fragments. Cells were further cultured in Dulbecco’s modiﬁed Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), pen- icillin (100 lg/mL), and streptomycin (100 lg/mL), in 5% CO2 chamber. Incubation of SMC with the Coated MNPs All the coated MNPs was sterilized with ﬁlter-ﬁlm (pore size, 0.22 lm) and sonicated before dilution into DMEM culture medium to ensure even particle suspension. Then, the MNPs were diluted with DMEM at different concen- tration and added into the plates in triplicate for a further speciﬁed time after the normal medium was removed. All control cells were cultured in the absence of any particles. Every experiment was repeated at least three times independently. Cellular Uptake of MNPs Assay Cellular uptake of MNPs was evaluated according to the method of Petri-Fink [26]. The supernate of cells on the 6- well plates was removed and cells were thoroughly washed with PBS and resuspended in 2 M HCl (1 mL/well of a 6- well plate) at 37 C for 2 h. The protein concentration of 123