The human epithelial cell line A549 was obtained from Merck KGaA, Darmstadt, Germany. Cells were cultivated at 37°C in a humidified atmosphere with 5% CO₂ in RPMI-1640 medium with 10% fetal bovine serum (Merck KGaA), 100 units ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin. The microparticles were produced from amorphous silica nanoparticles (Polysciences GmbH, Eppelheim, Germany) as described previously (2) and had a length of 10 µm and diameter of 3 µm. The microparticles were suspended in aqua dest distilled water, and the number of particles per µl were determined by the use of a Neubauer counting chamber. A total of 2×10⁶ particles were incubated with 1×10⁵ cells to establish a ratio of 20:1. The incubation times with the microparticles were 3, 6, 8, 24 and 48 h.

The living cells incubated with microrods were analysed by fluorescence microscopy (Carl Zeiss AG, Oberkochen, Germany) and confocal laser scanning microscopy (VTinfinity, VisiTech, UK), as previously described for mouse cardiac ventricular myocytes (5). Cell borders were labelled using the membrane dye CellMask Deep Red according to the instructions of the supplier (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All microscopy examinations were performed at room temperature (20–22°C).

For an MTT assay, A549 cells were cultured in 96-well plates with 200 µl RPMI-1640 medium per well. Each well contained 1×10⁴ cells and 0 (control), 1×10⁴ or 1×10⁵ microrods. Following incubation for 1, 3, 6 and 24 h the cells were washed once with sterile phosphate-buffered saline (PBS). The cells were then incubated in a humidified atmosphere of 5% CO₂, 95% air at 37°C with 5 mg/ml MTT (Merck KGaA) dissolved in PBS buffer at 1:10 ratio under gentle shaking for 4 h. Removal of the MTT reagent was followed by the addition of 100 µl dimethyl sulphoxide and incubation for 20 min in the humidified atmosphere of 5% CO₂, 95% air at 37°C to dissolve the formazan crystals. To determine cell viability, the absorbance at 550 nm was measured using a microplate spectrophotometer. For each concentration and time point, triplets were used.

For freeze-fracture analysis, A549 cells were cultured on poly-L-lysine coated glass coverslips. The confluent layer was fixed with 2% paraformaldehyde at room temperature for 5 min. Incubation in 30% glycerol in Soerensen's phosphate buffer (0.15 M, pH 7.4) as pre-vitrificational cryoprotection was performed at 4°C for 30 min. Small pieces of the glass coverslips were mounted headfirst onto the flat top of copper specimen carriers (Baltic-preparation, Niesgrau, Germany) and subsequently plunge-frozen in nitrogen-cooled liquid ethane using a cryopreparation chamber (Leica Microsystems GmbH, Wetzlar, Germany). Frozen samples were mounted onto a nitrogen-cooled double replica table and inserted into a BAF060 freeze-fracture device (Leica Microsystems GmbH). Freeze-fracturing was performed by cracking the double carrier open at −162°C and 1×10⁻⁷ mbar pressure. Fractured samples were coated with a 1-nm pre-carbon coat applied at a shadowing angle of 90°, a 1-nm platinum-carbon coat applied at 60° and a second carbon coat of 20 nm applied at 90° (carbon coat: carbon rods 3×50 mm, carbon/platinum coat: carbon rods 2×20 mm, platinum inlets 1.5×2 mm; Leica Microsystems GmbH). The frozen replicas were stabilized on a gold index grid (Plano GmbH, Wetzlar, Germany) using a drop of 0.5% Lexan polycarbonate plastic dissolved in dichloroethane (DCE; Acros Organics; Thermo Fisher Scientific, Inc.) (6). To evaporate the DCE and consolidate the Lexan, the assembly of carrier-sample-replica-Lexan-grid was incubated at −20°C for 16 h. Subsequently, the samples were thawed at room temperature and the carriers were removed. Digestion of the cell samples was performed under agitation in SDS-digestion buffer (2.5% SDS, 10 mM Tris-HCl, pH 8.9) at 60°C for 27 h. Prior to electron microscopy analysis, the Lexan film was resolved and removed by dipping the replica in DCE. Analysis was performed in a FEI Technai G2 transmission electron microscope (Thermo Fisher Scientific, Inc.) at 100 kV. Pictures were taken with an 8-bit camera at an image size of 1.42 megapixels (Olympus MegaView III; Olympus Soft Imaging Solutions GmbH, Münster, Germany).

It was uncertain whether the microrods are able to enter epithelial cells. The A549 cell line used is established as an epithelial cell line, which was supported by the freeze-fracture analysis (Fig. 2). In this representative image, distinct tight junctions are apparent.

From the MTT assay, cell viability at a 1:1 ratio of cells to microparticles was 101.9±0.9% after 1 h, 99.9±3.7% after 3 h, 103.6±1.5% after 6 h, 95.9±5.6% after 24 h, and at a 10:1 ratio was 103.7±5.1% after 1 h, 94.0±3.2% after 3 h, 90.3±2.5% after 6 h and 83.6±2.1% after 24 h (data not shown).

The fluorescence microscopy provided visualisation of the epithelial cells following co-incubation with the microrods at different time points (Fig. 3). After 8 h, microparticles remained extracellular (Fig. 3A). After 24 h, the majority of the rods had started to disaggregate (Fig. 3B). After 48 h, nearly all of the rods appeared to have disaggregated, and nanoparticles or conglomerates of nanoparticles appeared to be intracellular (Fig. 3C). Through confocal analysis, this process could be observed in greater detail and higher resolution (Fig. 4). After 1 h, attachments of the rods to the cells were observed (Fig. 4A and B). After 3 and 6 h (Fig. 4C and D), disaggregated nanoparticles were observed within the cells. After 48 h (Fig. 4E and F), many epithelial cells contained nanoparticles.