Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry

Background The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. Results Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells. Conclusion The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs.


Cytotoxicity of SiO 2 nanoparticles (NPs)
To determine non cytotoxic concentrations of NPs to be used for experiments, the metabolic activity of the cells was assessed by the WST-1 assay. Cells were seeded in 96-well plates at 10,000 cells per well in complete culture medium and incubated for 48 h before treatment with 100 µl of NPs at concentrations from 2.5 to 80 µg/cm² for 24 h. Metabolic activity was assessed using the WST-1 cell proliferation reagent (Roche, Meylan, France) according to the manufacturer's recommendations. After 4 h of incubation with cells, supernatants were transferred to a fresh plate in order to decrease the potential interference of NPs during the absorbance measurement. The absorbance was measured by dual wavelength spectrophotometry S3 at 450 and 630 nm using a microplate reader. Results are shown in Supporting Figure S1A. for 50 nm-FITC-SiO 2 NPs and S1B. for 100 nm-Por-SiO 2 .
Supporting Figure S1. Cell viability assay of NCI-H292 cells treated with NPs. Cells were exposed for 24 h to SiO 2 NPs of 50 nm (A.) and 100 nm (B.) size at different concentrations.
Metabolic activity was assessed by the WST-1 assay. The data were normalized to control values (no NP exposure) and expressed as mean ± SEM, n = 6 each. Data were analyzed by ANOVA, followed by Bonferroni post hoc test. * are indicating that there is a statistical difference compared to the control, p < 0.05.

Quenching of intracellular FITC fluorescence with Trypan Blue
Trypan Blue (TB) is a vital stain that enters only dead cells. When incubated with living cells it stays at the cell surface and should not be able to quench the fluorescence that is located inside S4 the cells. We assessed the efficiency of TB to quench only the fluorescence that is in direct contact by comparing the Median Fluorescence Intensity (MFI) of cells stained with 3,3′-Dihexyloxacarbocyanine iodide (DiOC 6 (3), Sigma), a mitochondrial marker, with and without TB. Cells were seeded in 12-well plates in complete cell culture medium at 10,000 cells/cm² for 48 h and then washed with Phosphate Buffered Saline (PBS, Life Technologies) and harvested by incubation with 0.05% Trypsin-EDTA for 10 min whose action was stopped by 10% FCS. The cell suspension was centrifuged for 5 min at 200 g then resuspended in media containing 1nM DiOC 6 (3) and incubated at 37°C in the dark for 30 min. Shortly before FCM analysis, cells were incubated with 0.11% Trypan Blue (Sigma) for 1 min. Cell-associated fluorescence was detected using a CyAn ADP LX (Dako Cytomation, Beckman Coulter, Villepinte, France) flow cytometer as described in methods section for 50 nm-FITC-SiO 2 -NP uptake quantification. Intracellular fluorescence remains the same when the cells were treated with 0.11% TB.
(Supporting Figure S2). Figure S2. Detection of DioC green fluorescence in viable cells in presence or not of Trypan Blue by FCM. Cells were incubated for 30 min with DioC. Shortly before FCM S5 analysis, cells were incubated or not with 0.11% TB. MFI is determined for 10,000 cells. The data are expressed as mean ± SD, n = 3 each.

Confocal images of the cells treated with 100 nm-Por-SiO 2 NPs at 4°C
Cells were treated with 100 nm-Por-SiO 2 NPs for 4 h at 25 µg/cm² at 4°C in order to block the active as well as passive diffusion of NPs inside the cells and to verify the adsorption of NPs on the cellular membrane. Staining with phalloidin has been performed as described in the method section. After the treatment NPs were observed exclusively outside the cells and as a layer firmly attached to the cellular surface (Supporting Figure S3).

Uptake of 50 nm-FITC-SiO 2 NPs studied by Imagestream
In order to compare the Imagestream technique with TB quenching using classical flow cytometry we analyzed values of MFI of the total cell and after applying the mask eroded for 6 pixels to compare them with results obtained with and without TB of Figure 2. 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 50 nm-FITC-SiO 2 -NPs at 2.5 and 5µg/cm² for 4 and 24 h.
Cells were prepared for analysis as described in the method section. Camera magnification was 40 x, a 488 nm excitation laser at 20 mW and a 785 nm excitation laser at 2.33 mW were used.
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. We observed that the values inside the eroded mask were lower than the ones for the total cell for both tested concentrations and had very similar tendency as the values obtained by flow cytometry before and after adding TB (Supporting Figure S4).

Mechanism of action of pharmacological inhibitors
Three endocytotic pathways were intensively investigated: clathrin dependent endocytosis, caveolae dependent endocytosis and macropinocytosis. 1 For each of the three main endocytotic pathways we used two pharmacological inhibitors considered as specific. The mechanism of action of these inhibitors is listed in the Supporting Table 1.  Table 1. Mechanism of action of pharmacological inhibitors of main endocytotic pathways.

Cytotoxicity of metabolic inhibitors
The

Preservation of actin filaments after treatment with pharmacological inhibitors
The effect of pharmacological inhibitors on the actin filaments was verified by confocal microscopy. Cells were treated with inhibitors for 4 h at concentrations used in experiments before staining of actin filaments with phalloidin. Experiments have been performed as described in the method section.
Confocal microscopy revealed that actin filaments were intact after the treatment with inhibitors (Supporting Figure S6).

S18
To determine non cytotoxic concentrations of TiO 2 NPs to be used in experiments, the metabolic activity of the cells was asessed by WST-1 assay. Cells were seeded in 96-well plates at 10,000 cells per well in complete cell culture medium and incubated for 48 h before treatment with 100 µl of NPs from 5 to 80 µg/cm² for 24 h. Metabolic activity was assessed as already described. TiO 2 NPs did not impair the metabolic activity of the treated cells at any concentration tested (Supporting Figure S10). were normalized to control values (no NP exposure) and expressed as mean ± SEM, n = 6 each.

NP synthesis
S19 50 nm-FITC-SiO 2 -NPs were synthesized following a slightly modified method described by Van Blaaderen to obtain the expected size 12  The experiment is carried out as follows: the entire mixture was poured into a 1 L round bottom flask containing 500 mL ethanol, 160 mL water and 9.9 mL ammonium hydroxide (28%). Then 18.2 mL of TEOS was added twice spaced 12 h apart to prevent secondary nucleation. The resulting particles have an approximate average diameter of 50 nm measured from TEM images.
After the synthesis, ammonia and ethanol were removed from the medium by rotary evaporation at 40°C. The fluorescent particles were extensively washed by centrifugation against ultrapure water (18 M ) at 13,000 g for 15 min until disappearance of fluorescence in the supernatant.
Estimation of silica concentration in the dispersion was carried out by inductively coupled plasma optical emission spectrometry (ICP-OES) and gravimetric method.

Dynamic Light Scattering (DLS) analysis
NPs were characterized for their hydrodynamic diameter and zeta potential whilst suspended in RPMI. DLS and zeta potential values were measured by a Zetasizer (nano ZS, Malvern Instruments, USA, Supplementary