3-aminopropyltrimethoxysilane (APTS) coated Fe3O4 NPs were synthesized using hydrothermal approach as described previously21. Briefly, 1 g FeCl2·4H2O was dissolved in 6.2 ml distilled water, then 5 ml ammonium hydroxide was added and the suspension was continuously stirred at room temperature for 10 min. Then, 2 ml APTS was added and the mixture was autoclaved at 134℃ for 3 h. Then the mixture was cooled and purified with distilled water, then centrifuged (5,000 rpm, 10 min) to remove excess reactants. The process was repeated for five times to get APTS-coated Fe3O4 NPs.
The amine groups were connected on the surface of APTS-coated Fe3O4 NPs via a reaction with acetic anhydride. In brief, 6 mg APTS-coated Fe3O4 NPs was dispersed in 5 ml ethanol and mixed with 1 ml of triethylamine. 5 ml dimethyl sulfoxide (DMSO) containing 1 ml acetic anhydride was then added and mixed for 24 h. Then the mixture was purified with distilled water, then centrifuged to remove excess reactants. The process was repeated for three times to get acetylated APTS-coated Fe3O4 NPs.
Characterization of NPs
The morphology of NPs was observed under JEO2010 F transmission electron microscope (Akishima-shi, Japan) operated at 200 kV. The size of NPs was measured using Image J image analysis software. The size distribution histogram was obtained via different TEM images. Fe concentration of APTS-coated Fe3O4 NPs was tested with Prodigy inductively coupled plasma-atomic emission spectroscopy (ICP-AES) (PerkinElmer Optima 8000, USA).
The intracellular uptake of NPs
The intracellular uptake of APTS-coated Fe3O4 NPs was quantified via ICP-AES analysis with Prodigy ICP-AES system (PerkinElmer Optima 8000, USA). U87 cells were seeded in six-well plates at density of 1×106 per well and cultured for 24 h, then incubated with different concentrations of acetylated APTS-coated Fe3O4 NPs (0, 10, 25, 50, and 100 μg/ml) for 24 h or incubated with 25 μg/ml acetylated APTS-coated Fe3O4 NPs for 6, 12, 24, 48 or 72 h. The culture medium was removed and the cells were washed with PBS three times. The cells were collected and lysed. The amount of iron in the lysates was quantified with ICP-AES.
The viability of U87 glioma cells was tested via MTT assay as described previously22. Briefly, U87 cells were seeded into a 96-well plate with 1×104 per well and incubated for 24 h. Next the cells were incubated with different concentrations of acetylated APTS-coated Fe3O4 NPs (0, 1, 10, 20, 40, and 80 μg/ml) for another 24 h, then treated with or without irradiated with different dose of X-ray (0,2,4,6,8,10 Gy) and cultured for 24 h. 20 μl of 5 mg/ml MTT solution was added to each well. After 4 h of incubation, the medium was removed and 200 μl DMSO was added. The absorbance was measured in a BioTek Elx800 (Thermo Scientific, Waltham, MA, USA) at a wavelength of 490 nm. The inhibition of cell growth was calculated with following formula: Cell viability (%) = (mean of Abs. value of treatment group/mean of Abs. value of control group)×100%. All treatments were carried out in triplicate.
Apoptotic cells were detected by double-staining with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). U87 cells were treated with APTS-coated Fe3O4 NPs or/and X-rays as described above. The cells were collected and resuspended in Annexin V binding buffer, then stained by Annexin V-FITC and PI according to the protocol of Annexin V-FITC Apoptosis Detection Kit (Sigma). Analysis of stained cells was performed in triplicate with each sample of 20,000 cells by a FACSCalibur flow cytometer (Becton–Dickinson, BD Biosciences, Ontario, Canada).
Cell cycle analysis
U87 cells were treated with APTS-coated Fe3O4 NPs or/and X-rays as described above. The cells were collected and resuspended in DNA staining solution (Multiscience, China) containing 10 μg/ml RNase and 50 μg/ml PI, then stained by PI for 30 min at room temperature in the dark. Analysis of stained cells was performed in triplicate with each sample of 20,000 cells by a FACSCalibur flow cytometer (Becton–Dickinson, BD Biosciences, Ontario, Canada). The data were analyzed by ModFit LT software.
Values were expressed as the mean ± standard deviation (SD) and analyzed by SPSS version 13.0. Statistical analysis was carried out using t test or χ2 test. P<0.05 was considered significant.