The ultrastructure of HCT-116 and A549 cells under different incubation conditions

Many of the above-mentioned conclusions drawn about the processing of the nanoparticles based on the hard corona proteome result in characteristic ultrastructural changes in the living cells. These ultrastructural changes induced by the internalized gold nanoparticles, as well as their intracellular accumulation sites can elucidate the cellular response mechanisms to the internalization of the gold nanoparticles. To obtain ultrastructural information, cryo soft X-ray nanotomography (XRT) experiments were performed, which yielded three-dimensional visual information about organelles and internalized gold nanoparticles in their quasi-native state preserved by vitrification.

Both cell lines were cultured in DMEM-FBS and McCoy-FBS media as well to see the possible effects of the choice of culture medium. DMEM-FBS is one of the most common cell culture media nowadays, and numerous studies use it in experiments with HCT-116 [283-285], while the distributor ATCC^® suggests the use of McCoy-FBS in their cultivation. McCoy’s Modified 5A medium is more supplemented than Dulbecco’s Modified Eagle’s Medium [286], and as a possible result of improper nutrition, HCT-116 cells grew slower in DMEM-FBS than in McCoy-FBS as observed under a light microscope. Gold nanoparticles were added to the culture medium of choice in non-aggregated and in non-aggregated form, respectively, to elucidate the influence of pre-aggregation on the internalization and the intracellular aggregate morphology (pre-aggregation was achieved with a final NaCl concentration of 0.1 M). Since the resolution of the reconstructed tomograms is ~9.8 nm/pixel, 30 nm gold nanoparticles are visible even as single particles in the intracellular compartments. The tomograms were reconstructed in Etomo using the internalized gold nanoparticles as fiducial markers.

Tomographic slices of HCT-116 cells incubated with non-aggregated gold nanoparticles in DMEM-FBS and in McCoy-FBS, respectively, are shown in Figure 9.2. In both cell culture media, gold nanoparticles are aggregated inside the cells, which suggests that gold nanoparticles are actively processed. Since signs of clathrin-mediated endocytosis were found in the hard corona proteome (Table S9.1), which allows the uptake of only a few nanoparticles in the same vesicle, it can be concluded that the aggregate formation occurred inside the cells. Several different traits can be observed in the two tomographic

121 slices representing the incubation of cells in DMEM-FBS and McCoy-FBS media, respectively.

Figure 9.2. Tomographic slices of HCT-116 cells grown in DMEM-FBS (A) and McCoy-FBS (B) incubated with non-aggregated gold nanoparticles. The images are representative of 11 and 14 tomograms, respectively. Mitochondrion: M, liposome: L, plasma membrane: PM, nuclear membrane: NM, nucleus: Nu, nucleolus: ns, vesicles: V, gold nanoparticles are marked with red arrowheads. The scale bars represent 2 µm.

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The increased number of small, round mitochondria suggests mitochondrial fission (Figure 9.2A), and the dilated perinuclear cisternae (Figure 9.2A) indicate that apoptosis could be initiated in HCT-116 cells cultured in DMEM-FBS [287-290] after internalizing non-aggregated gold nanoparticles. In contrast, when HCT-116 cells were grown in McCoy-FBS and incubated with non-aggregated gold nanoparticles (Figure 9.2B), longer mitochondria and intact nuclear membrane were observed. Therefore, it is possible that the use of DMEM-FBS promotes cellular stress by the improper nutrition of the HCT-116 cells: essential components of the McCoy-FBS, e.g., biotin, vitamin B12, or ascorbic acid are not constituents of the DMEM-FBS medium. Furthermore, the lack of choline has been shown to promote apoptosis [291], which has a three-times lower concentration in DMEM-FBS than in McCoy-FBS. It is known that gold nanoparticles can trigger mitochondrial damage, which results in mitochondrial fission or apoptosis [282].

However, the smaller mitochondria could still be active, since the mitochondrial cristae are well contoured, suggesting that these organelles are intact. The signs of the cellular and organelle damage due to the uptake of gold nanoparticles can be more prominent in the cells cultured in DMEM-FBS as a result of the processes promoting the survival of cells being less efficient under less optimal culture conditions.

Contrary to the incubation with non-aggregated nanoparticles, HCT-116 cells show traits of cellular injury and stress in both culture media upon incubation with pre-aggregated nanoparticles (Figure 9.3). Mitochondrial fission (not seen in the figure) or hyperfusion (Figure 9.3A) can be observed in the whole tomogram due to mitochondrial damage and higher energy demand [289, 292], respectively. In the nucleus, chromatin tends to form granules (Figure 9.3), and large vacuoles with bright lumen appear in the cytoplasm (Figure 9.3B), which indicate cellular damage [293-295]. It can be excluded that the granules in the nucleus are the naturally abundant heterochromatin patches, since they are present in every living cell, yet the granules seen in Figure 9.3 are unique compared to Figure 9.2. The darkness of the granules, that is, their higher X-ray absorption relative to their surroundings means that they are tightly packed, which is a result of the possibly occurring chromatin condensation [295].

123 Figure 9.3. Tomographic slices of HCT-116 cell incubated with pre-aggregated gold nanoparticles in DMEM-FBS (A) and in McCoy-FBS (B). The images are representative of 12 and 15 tomograms, respectively. Mitochondrion: M, liposome: L, plasma membrane: PM, nuclear membrane: NM, nucleus: Nu, vesicles: V, gold nanoparticles are marked with red arrowheads. The scale bars represent 2 µm.

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The virtual segmentation of the whole tomogram (Figure 9.4) corresponding to the tomographic slice in Figure 9.3B visualizes the chromatin granules (blue volumes) and the high intracellular volume occupied by the cytoplasmic vacuoles (red volumes). It has been shown that the excessive endocytosis of gold nanoparticles can induce apoptosis and autophagy [282], the latter resulting in the accumulation of large cytoplasmic vacuoles similar to those shown in Figure 9.3B. The cytoplasmic vacuoles with bright lumen can also originate from the dilation of the endoplasmic reticula [293] or other processes related to the water permeation of the cell membrane [294], which all suggest cellular injury or cell death.

Figure 9.4. The segmentation of the tomogram represented by a tomographic slice in Figure 9.3B (HCT-116 cell, McCoy-FBS, pre-aggregated gold nanoparticles). Red volumes: vacuoles, yellow volumes: gold nanoparticles, green mesh: nuclear membrane, blue volumes: chromatin granules.

To learn whether the same ultrastructural changes can be induced in A549 as well, they were cultivated identically in both culture media like HCT-116 cells (Figures 9.5 and 9.6).

The growth rates of A549 cells in the two culture media were comparable.

125 Figure 9.5. Tomographic slices of A549 cells incubated with non-aggregated (A) and pre-aggregated (B) gold nanoparticles in DMEM-FBS. The images are representative of 14 and 6 tomograms, respectively. Mitochondrion: M, liposome: L, plasma membrane:

PM, nuclear membrane: NM, nucleus: Nu, vesicles: V, gold nanoparticles are marked with red arrowheads. The scale bars represent 2 µm.

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Figure 9.6. Tomographic slices of A549 cells incubated with non-aggregated (A) and pre-aggregated gold nanoparticles (B) in McCoy-FBS. The images are representative of 11 and 12 tomograms, respectively. Mitochondrion: M, liposome: L, plasma membrane:

PM, nuclear membrane: NM, nucleus: Nu, vesicles: V, gold nanoparticles are marked with red arrowheads, apoptotic bodies: AB. The scale bars represent 2 µm.

127 The ultrastructural features of A549 cells after their incubation with non-aggregated gold nanoparticles are shown in Figure 9.5A and Figure 9.6A. In both cell culture media, A549 cells appeared healthy, though, in Figure 9.5A representing A549 cells grown in DMEM-FBS and incubated with non-aggregated gold nanoparticles, mitochondria appear to be slightly more abundant in the whole tomogram than in the cells grown in McCoy-FBS (Figure 9.6A). It can be seen that the gold nanoparticles form intracellular aggregates of a few nanoparticles. When A549 cells are incubated with pre-aggregated gold nanoparticles (Figure 9.5B in DMEM-FBS and Figure 9.6B in McCoy-FBS), the signs of cellular injury and cellular stress appear similar to those observed in HCT-116 cells (compare with Figures 9.2 and 9.3). The altered mitochondrial morphologies (rounding due to fission or long, branching structures as a result of hyperfusion [292]) infer changes in the energy production and energy demand of the cells.

Chromatin granules formed in the nucleus (Figure 9.6B) and the disruption of the nuclear membrane (Figure 9.5B and Figure 9.6B) both reveal the presence of cellular damage or cell death as discussed in the case of HCT-116. The formation of extracellular vesicles can be observed in Figure 9.6B, which are probably apoptotic bodies according to the mass spectrometric results (Table S9.2), thus exhibiting a clear sign of induced apoptosis (Figure 9.6B). It can be concluded that the XRT experiments yielded valuable information about the cellular stress and injury induced by gold nanoparticles on a single-cell level. The extent of the ultrastructural changes implying single-cellular injury and single-cell death depends both on the cell line and the chosen incubation conditions.