Fluorescent SiO2 NPs of two sizes (50 and 100 nm) were synthesized. Fluorochromes were incorporated inside the NP core during synthesis. 50 nm-FITC-SiO2-NPs were labeled with fluorescein isothiocyanate (FITC) that is characterized by an excitation wavelength at 488 nm and maximum emission wavelength at 520 nm. The NPs were synthesized as described in Supporting Information (section 12). 100 nm-Por-SiO2-NPs were synthesised as already described
. They were coupled with 5, 10, 15, 20 -Tetrakis-(1-methyl-4pyridino) porphyrine tetra (toluene-4-sulfonate) or shortly porphyrine characterized by an excitation wavelength at 422 nm and maximum emission wavelengths at 666 nm and 716 nm. Non fluorescent TiO2 NPs of 10 nm size with neutral, positive and negative surface charges were synthesized as described previously
Nanomaterial characterization (TEM, DLS, fluorescence excitation/emission spectrums)
Morphology and aggregation of SiO2 NPs were verified by Transmission Electron Microscopy (TEM, JEOL 1200 EXII (OXFORD LINK ISIS 300)) as well as by Dynamic Light Scattering (DLS) analysis in RPMI 1640 cell culture medium (Life Technologies). For DLS characterization, NPs were prepared at the highest concentrations used in experiments. DLS values as well as the values of zeta potential were measured by Zetasizer (nano ZS, Malvern Instruments, USA). NP excitation/emission spectrums were verified by confocal laser scanning microscope (Zeiss 710 confocal microscope). All the results are presented in the Supporting Information (sections 13, 14 and 15). Non fluorescent TiO2 NPs of 10 nm size were characterized as described previously
Human lung adenocarcinoma (NCI-H292) cells were purchased from the American Type Culture Collection (Sigma-Aldrich, Saint Quentin Fallavier, France) and grown in RPMI 1640 (Roswell Park Memorial Institute) medium without phenol red (Life Technologies), containing 10% fetal calf serum (FCS, Life Technologies) and 1% glutaMAXTM (Life Technologies), subsequently referred to as complete cell culture medium. The NCI-H292 cell line was derived from a lymph node metastasis of a pulmonary mucoepidermoid carcinoma. All experiments were performed with cells from passages 13 to 20. Cells were grown in T75-flasks (Costar, Sigma) as a monolayer. Exponentially growing cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37°C and were passaged twice weekly using 0.05% Trypsin-EDTA (Life Technologies) whose action was stopped with 10% FCS.
Depending on experiment, cells were seeded into cell culture plates (Costar, Sigma) and treated when they had reached 70-80% confluence. Before NP exposure, cells were rinsed with PBS to eliminate trace amounts of FCS. Treatments were performed in the absence of FCS as it has been found previously that the serum can form a protein corona modulating NP uptake and as the bronchial cells are not directly exposed to serum proteins conversely to internal organs. 50 nm-FITC-SiO2-NP stock (23.2 mg/mL in water) was vortexed shortly before making final dilution for the treatment. 100 nm-Por-SiO2-NPs stock suspension (5.25 mg/mL in water) was sonicated in an ultrasonic bath (Branson Cleaner, Ultrasonic, B200) at 20 W for 10 min then vortexed before making dilutions for treatment. TiO2 NPs stock suspension (2.56 mg/mL in water) was sonicated at 60 W (Ultrasonic processor, Bioblock Scientific) for 8 min. Cells were exposed for different times with either FITC labeled SiO2 NPs at 2.5 and 5 μg/cm2, porphyrine-labeled SiO2 NPs at 5, 15 and 25 μg/cm2 or non fluorescent TiO2 NPs at 20 and 40 μg/cm2.
For endocytic inhibition experiments, cells were pre-incubated for 30 min with different endocytic inhibitors at the following concentrations: chlorpromazine (CP) 25 μM, monodansylcadaverine (MDC) 75 μM, EIPA 75 μM, amiloride (A) 1.5 mM, nystatine (N) 75 μM and filipine (F) 4.5 μM (all from Sigma). Energy dependence experiments were performed by pre-incubating the cells at 4°C or with sodium azide (NaN3, 100 mM, Sigma) for 30 min prior to exposure to NPs. After these pre-incubations, NPs were added to cell cultures and incubated for 3.5 h, either in the presence of drugs or at 4°C.
Flow cytometry (FCM) analysis
Cells were seeded in 12-well plates at 10,000 cells/cm2 in complete cell culture medium and incubated for 48 h before treatment. After treatment with 1.2 mL/well of 50 nm-FITC-SiO2-NPs at indicated concentrations, medium was removed, cultures were thoroughly washed with PBS and cells were harvested by trypsination whose action was stopped with 10% FCS. Shortly before FCM analysis cells were incubated with 0.11% Trypan Blue for 1 min in order to quench the FITC-fluorescent signal coming from NPs adsorbed to the cell surface. Cell-associated fluorescence was detected using a CyAn ADP LX (Dako Cytomation, Beckman Coulter, Villepinte, France) flow cytometer. Laser excitation and emission bandpass wavelengths were 488 nm and 575 ± 25nm respectively. Minimum of 10,000 cells was analyzed after exclusion of the cellular debris from the analysis by gating on the 575 nm Log versus FS area graph. The results are reported as the median of the distribution of cell fluorescence intensity obtained by analyzing 10,000 cells in the gate.
Imaging flow cytometry analysis
Cells were seeded in 6-well plates at 10,000 cells/cm2 in complete cell culture medium and incubated for 48 h before treatment with 2.9 mL/well of 100 nm-Por-SiO2-NPs at indicated concentrations. At the end of the exposure to NPs, the media was removed, cells were thoroughly washed with PBS and cells were harvested. Cell suspension was centrifuged for 5 min at 200 g at 4°C and the cell pellet was resuspended in 500 μl of 4% para-formaldehyde (PFA, Santa Cruz Biotechnology Inc, Heidelberg, Germany). After 20 min of incubation in PFA, cells were rinsed three times with PBS and finally suspended in at least 50 μl of PBS. At least 2,500 cells were analyzed using Amnis ImageStreamx platform (Amnis, Proteigene, Saint Marcel, France) and InspireTM system software (Amnis). Camera magnification was 40x, 488 nm excitation laser was at 100 mW and 785 nm excitation laser was at 2.33 mW, except for non fluorescent NPs where it was set at 0.01 mW. The images were acquired with a normal depth of field, providing a cross-sectional image of the cell with a 4 μm depth of focus. A mask representing the whole cell was defined by the bright-field image, and an internal mask was defined by eroding the whole cell mask by 6 pixels (equivalent to 3 μm, as the size of 1 pixel is 0.5 μm) in order to eliminate the fluorescent signal coming from the NPs attached to the cell surface thus measuring only the internalized part. The results are analyzed by IDEAS software (Amnis),. Values of the internalization score, mean fluorescence intensity and mean side scatter intensity were calculated for at least 500 cells per sample.
Cells were seeded in 8 well Lab-TekTM chambered coverglasses (Nunc, Thermo Scientific, Dominique Dutscher, Brumath, France) at 40,000 cells/well in complete cell culture media. After treatment with 0.22 mL/well of NPs at indicated concentrations, cells were fixed in 4% PFA for 20 min at 25°C, rinsed 3 times with PBS and incubated for 10 min with NH4Cl (50 mM, Sigma) before permeabilization in 0.05% PBS-Tween20 (Sigma). To stain the actin filaments, cells were incubated for 30 min with FITC or TRITC-phalloïdine (0.9 nM in PBS,), then rinsed 4 times in PBS. Cells were mounted in Polyvinyl alcohol mounting medium with DABCO® (Sigma).
For immunolabelling experiments, cells were fixed with methanol at −20°C for at least 20 min, and rinsed three times with PBS. After permeabilization in 0.05% PBS-Tween20 and saturation in 0.01% PBS-Tween20–3% Bovine Serum Albumin (BSA, Sigma) cells were incubated for 60 min with goat polyclonal clathrin heavy chain antibody, C-20 (sc-6579, 1:50, Santa Cruz), goat polyclonal SNX5 D-18 (sc-10625, 1:50, Santa Cruz) or rabbit polyclonal caveolin1 N-20 (sc-894, 1:50, Santa Cruz) antibodies in 0.01% PBS-Tween20-3% BSA. Secondary anti goat antibodies Alexa fluor 488-IgG or 647-IgG (Life Technologies), anti rabbit antibodies Alexa fluor 488-IgG or 546-IgG (Life Technologies) were diluted in 0.01% PBS-Tween20-3% BSA at 1:400 and incubated for 45 min. Cell nuclei were stained with DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride, Sigma, 0.25 μg/mL in PBS) for 1 min. Cells were examined under a Zeiss 710 confocal microscope using 63x objective (NA of 1.4) and a 1 to 1.5x zoom. The refractive index of immersion oil was 1.512. Considering optical laws the theoretical resolution was calculated and instrument settings adapted to obtain the best possible resolution in our images. Image treatment was done with Image J software (Image J 1.42 NIH, USA). The three-dimensional (3D) structure of the cells treated with NPs was reconstructed from corresponding confocal images using IMARIS software 7.5 (Bitplane). Pearson’s correlation coefficient was calculated using JACoP (Just Another Colocalization Plugin) for images with lateral resolution of 0.09 μm and axial resolution of 0.5μm.
siRNA knockdown experiments
The expression of clathrin heavy chain (CHC) protein in NCI-H292 cells was knocked down by transfection with specific siRNA targeted against this protein (SI00299880, Qiagen, Courtaboeuf, France). Cells were seeded in 6-well plates at 15,000 cells/cm2 in complete culture medium. 24 h after seeding cells were treated with 100 nM siRNA-control or siRNA-clathrin heavy chain using 18 μl/well of Hiperfect transfection reagent (Qiagen) as indicated in the transfection protocol provided by the supplier. Cells were retransfected every 24 h until 72 h when transfection efficiency was monitored by confocal microscopy and Western blot. After treatment with siRNA, cells were incubated with NPs during 3.5 h.
Every experiment was repeated at least twice with duplicates or triplicates of each condition. Data are represented as means ± SD or SEM and were analyzed on commercially available software SigmaStat (version 3.0, Systat software Inc, San Jose, California, USA) analysis of variance (one-way ANOVA) followed by Bonferroni post hoc test for multiple comparisons with p < 0.05 (two tailed) considered as significant.