Calcined diatomite was obtained by DEREF S.p.A (Castiglione in Teverina, Viterbo, Italy). 3-aminopro-pyltriethoxysilane (APTES), H2SO4, and tetramethylrhodamine isothiocyanate (TRITC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from GIBCO (Carlsbad, CA, USA). HCl was purchased from Romil (Cambridge, UK). Absolute ethanol and H2O2 was purchased from Carlo Erba (Milan, Italy); HEPES powder was purchased from Promega (Madison, WI, USA).
Purification of diatomite powder
Five grams of crashed diatomite rocks were resuspended into 250 ml of absolute ethanol and sonicated for 5 h to break large aggregates. The dispersion was sieved through a nylon net filter with pore size of 41 μm, and then filtered with pore size of 0.45 μm (Millipore, Billerica, MA, USA). The diatomite nanopowder was purified to remove organic and inorganic impurities [9, 10]. The sample was centrifuged and the pellet treated with Piranha solution (2 M H2SO4, 10% H2O2) for 30 min at 80°C. Nanoparticle dispersion was centrifuged for 30 min at 21,500 × g, washed twice with distilled water, resuspended in 5 M HCl, and incubated over night at 80°C. DNPs were then centrifuged for 30 min at 21,500 × g and washed twice with distilled water to eliminate HCl residues.
Characterization of nanoparticles size
The size and zeta-potential measurements of purified diatomite nanoparticles dispersed in water (pH = 7) were performed before and after APTES functionalization by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with a He-Ne laser (633 nm, fixed scattering angle of 173°, 25°C).
Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were also used to investigate nanoparticles morphology. Briefly, in TEM analysis, purified diatomite nanoshells were characterized by placing a drop of suspension on a TEM copper grid with a lacy carbon film and then observed by a Jeol 1011 TEM (Peabody, MA, USA) at an accelerating voltage of 100 KV. For SEM characterization, diatomite samples were deposited on crystalline silicon substrates mounted on a double-faced conductive adhesive tape. Images were acquired at 5-kV accelerating voltage and 30-μm wide aperture.
The human lung epidermoid cancer cell line (H1355), obtained from American Type Tissue Collection (Rockville, MD, USA), was grown at 37°C with an atmosphere of 5% CO2, in RPMI 1640 (GIBCO) medium supplemented with 10% heat inactivated FBS (GIBCO), 100 U/mL penicillin, 100 mg/mL streptomycin, 1% l-glutamine.
Purified nanoparticles were amino-modified with a 5% (v/v) APTES solution in absolute ethanol [13, 14]. The APTES film formation was carried out for 1 h at room temperature with stirring in a dark condition. After this step, the sample was centrifuged for 30 min at 21,500 × g and supernatant discarded. The functionalized diatomite were washed twice with absolute ethanol and the collected pellet was incubated for 10 min at 100°C (curing process). Finally, the sample was washed twice with absolute ethanol and twice with 20 mM HEPES buffer pH 7.5.
Fourier-transform infrared spectroscopy
Chemical composition of APTES-functionalized diatomite nanoparticles was analyzed by Fourier-transform infrared (FTIR) spectroscopy. Spectra were recorded by a Thermo-Nicholet NEXUS Continuum XL (Thermo Scientific, Waltham, MA, USA) equipped with a microscope, at 2 cm−1 resolution on samples deposited on silicon chips (p-type, 0.003 ohm cm resistivity, <100 > oriented, 500-μm tick) of about 1 cm × 1 cm.
Nanopowder diatomite labeling
Diatomite labeling procedure was based on the use of an aminoreactive molecule, tetramethylrhodamine isothiocyanate. TRITC powder was solved in dimethyl sulfoxide (DMSO) and incubated with diatomite nanopowder in the presence of NaHCO3 0.1 M pH 8.7 with stirring for 1 h at room temperature in a dark condition. Subsequently, the sample was washed with distilled water to remove TRITC excess, until no fluorescence was revealed in the supernatant when analyzed by fluorescence microscopy. Labeled diatomite nanoparticles will be indicated as DNPs*.
H1355 cell line (20 × 103 cells/coverslip) was plated on 10-mm glass coverslips placed on the bottom of 24-well plate, allowed to attach for 24 h under normal cell culture conditions, and then incubated with increasing DNPs* concentration (5, 10, 15 μg/mL) for 24 h. As negative control, the last supernatant obtained from nanoparticles labeling procedure was added to the cells. Cell nuclei and membranes were then stained with Hoechst 33342 (Invitrogen, Carlslab, CA, USA) and WGA-Alexa Fluor 488, respectively. Images were acquired at × 63 magnification on a LSM710 confocal fluorescence microscope (Carl Zeiss Inc., Peabody, MA, USA) with the appropriate filters. Cell fluorescence intensity was analyzed by using ImageJ software (http://imagej.nih.gov/ij/).