diameter and length of the nanotubes were obtained from about a hundred nanotubes in twenty transmission electron microscopy (TEM) images. The fNTs were characterized with a Jeol JEM-2010 TEM operating at 200 keV and a fluorescence spectrometer.
Loading Plasmid DNA into the Silica Nanotubes: The plasmid DNA encoding green fluorescence protein (GFP) (8 lg, Clontech) was added to the transparent nanotube (tNT) or fNT solution (200 lL) and mixed by rocking for 24 h at 4 C. Unbound DNA was removed by washing with water several times. Finally, a CaCl 2 solution (2 lL, 2 M) was added to the DNA/nanotube complex solution, which was incubated for an additional 24 h. As a control, free DNA (8 lg) and tNTs without loaded DNA (200 lL) were treated independently with the CaCl 2 solution and incubated for 24 h under the same conditions as the DNA/nanotube complex.
GFP-Transfection Experiments: COS-7 cells were cultured in 10 cm dishes in Dulbecco's Modified Eagle Medium (DMEM) with 10 % fetal calf serum (FCS) in the presence of 1 % penicillinΒ±streptomycin. The cells were grown at 37 C in a CO 2 incubator and passaged every 2Β±3 days. For transfection experiments, the cells were seeded on 3 cm dishes for 24 h. The serum-containing DMEM was then removed from the dishes and replaced by 200 lL of the DNA/fNT complex. After 6 h, the residual DNA/fNT complex in the solution was removed by washing with phosphate-buffered saline (PBS) and replaced with fresh serum-containing DMEM. The cells were incubated continuously for 48 h. For contrast experiments, the cells were treated with free plasmid DNA or fNTs under the same conditions. For observation, the cells were washed with PBS and observed with an epifluorescence or laser-scanning confocal microscope. The number of cells was counted by flow cytometry (Partec).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay: Different amounts of fNTs (from 100Β±400 lL in 50 lL intervals) were added to COS-7 cells. After 48 h incubation, the MTT assay (Acros) was performed as described by the manufacturer. In brief, the serum-containing medium was replaced by the MTT solution (200 lL). After incubation in the MTT solution for 4 h, the cells were collected by centrifugation and treated with dimethyl sulfoxide (500 lL). The optical density at 570 nm was measured with a spectrometer (Bio-TEK).