Intracellular aggregate properties based on tomogram segmentation

With the virtual segmentation of the tomograms, the intracellular gold nanoparticle aggregate morphologies can be revealed, which sheds light on the cellular response induced by their uptake. Gold nanoparticle aggregates were rendered in the tomograms represented in Figures 9.2-9.6 based on their high X-ray absorption, as shown in refs. [144, 168]. The tomogram segmentation and particle rendering depend strongly on the contrast of the tomographic slices, which was manually adjusted to provide the best rendering of the nanoparticles and their aggregates.

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The segmented gold nanoparticle aggregates in HCT-116 and A549 cells are shown in Figure 9.7. It can be observed that the size of intracellular aggregates increased when the cells were incubated with pre-aggregated gold nanoparticles (Figure 9.7 right column) compared to the incubation with non-aggregated gold nanoparticles (Figure 9.7 left column). In HCT-116, the estimated number of total internalized nanoparticles rendered in the individual tomograms (Figures 9.2 and 9.3) changes from 10500 to 20000 and from 8500 to 9000 in DMEM-FBS and McCoy-FBS, respectively, when they were incubated with pre-aggregated gold nanoparticles instead of single particles. In A549, the numbers of intracellular gold nanoparticles in the individual tomograms (Figures 9.5 and 9.6) changed from 15000 to 33500 and from 15000 to 24500 in DMEM-FBS and in McCoy-FBS, respectively, when they were incubated with pre-aggregated gold nanoparticles instead of single particles. The overall increment in the nanoparticle uptake in both cell lines is the result of the higher number of simultaneously internalized particles in the form of aggregates compared to the case of single nanoparticle uptake. In HCT-116, the significantly higher intracellular gold nanoparticle number in DMEM-FBS can be explained by the suboptimal nutrition of cells in this medium. Since the gold nanoparticles are covered with the primary protein corona formed in the serum when they reach the cell membrane, it is possible that the cell recognizes them as a source of nutrients and thus internalizes them more readily than in McCoy-FBS where the conditions are optimal for the cells. As seen in Figures 9.5 and 9.6, the signs of cellular stress in A549 become more prominent in the case of incubation with pre-aggregated gold nanoparticles. While the numbers of rendered nanoparticles in the cells incubated with single gold nanoparticles were comparable in both culture media, they increased in DMEM-FBS by over 37% when incubated with pre-aggregated gold nanoparticles. Based on the XRT results and the corresponding numbers of intracellular gold nanoparticles, it was found that the nanoparticle uptake was higher under higher cellular stress.

It can be seen by comparing the intracellular gold nanoparticle structures after incubation with non-aggregated gold nanoparticles that a larger number of aggregates with more homogeneous size distribution are present inside A549 (Figure 9.7E and G) than in HCT-116 (Figure 9.7A and C). Since the nanoparticles were administered as single particles and not as aggregates, these figures yield important information about the intracellular processing of the nanoparticles.

129 In the comparison of the average number of nanoparticles per aggregate in the tomograms, systematic differences were found in the two cell lines, which further inform about the mechanisms induced by the nanoparticles.

Figure 9.7. Tomographic segmentation reveals gold nanoparticle aggregates in the previously presented tomograms (Figures 9.2-9.6): HCT-116 cells grown in DMEM-FBS with non-aggregated (A) and pre-aggregated gold nanoparticles (B), HCT-116 cells grown in McCoy-FBS incubated with non-aggregated (C) and pre-aggregated gold nanoparticles (D). A549 cells grown in DMEM-FBS incubated with non-aggregated (E) and pre-aggregated gold nanoparticles (F), and A549 cells grown in McCoy-FBS incubated with non-aggregated (G) and pre-aggregated gold nanoparticles (H).

The average numbers of nanoparticles per aggregate were comparable in HCT-116, ranging from 108 to 137 (Figure 9.7 A-D) regardless of whether the particles were administered as single or pre-aggregated gold nanoparticles. In A549, more substantial differences were found: the average number of particles changed from 66 to 242

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(Figure 9.7 E-F) and from 111 to 221 (Figure 9.7 G-H) in DMEM-FBS and McCoy-FBS, respectively, when the cells were incubated with pre-aggregated nanoparticles instead of single particles. Based on these numbers, HCT-116 readily processes the nanoparticles and their aggregates further after their uptake, thus resulting in comparable numbers of particles per intracellular aggregate in each sample, opposed to A549, which preserves the state of the internalized particles more. The difference between the nanoparticle processing of the two cell lines can be explained by comparing the hard corona proteomes with the intracellular aggregate morphologies. Based on the mass spectrometric results (Table S9.2), one of the most abundant proteins in the hard corona extracted from A549 is vimentin, which plays a key role in intracellular aggresome formation [281].

Aggresomes are the deposit sites of aggregated or misfolded proteins regulated by the cell. It has been known that the proteins forming the corona are what the cell “sees” [19];

however, these results indicate that A549 cells might see the nanoparticle corona as a form of aggregated proteins. The formation of a vimentin cage around the gold nanoparticles can reduce oxidative stress as it was previously shown [296]. This might explain why A549 cells with a high abundance of vimentin around the nanoparticles are more robust to the administration of gold nanoparticles than HCT-116 cells, which process the nanoparticles and their aggregates based on different mechanisms, as also indicated by the hard corona proteome (Table S9.1).

While the total number of aggregate structures increased in HCT-116 when the internalized particles were pre-aggregated (Figure 9.7 A-D), in A549 their number decreased (Figure 9.7 E-H). Based on the presence of related proteins in the hard corona, clathrin-mediated endocytosis is a possible uptake mechanism for the internalization of gold nanoparticles by HCT-116. However, clathrin-mediated endocytosis only explains the uptake of single particles or small aggregates formed by 2-6 particles [79-81]. Another mechanism has to be activated for the uptake of larger structures, e.g., macropinocytosis.

It has been shown by Palvai et al. that HCT-116 cells readily combine clathrin-mediated endocytosis with macropinocytosis during the uptake of nanoparticles [297]. The tomography segmentation data suggest that macropinocytosis is less abundant in A549 than in HCT-116, as the total number of intracellular aggregates decreases upon incubation with pre-aggregated gold nanoparticles.

131 According to previous discussions, macropinocytosis is not a prioritized uptake mechanism by A549 cells [84], which explains the decrement of intracellular gold nanoparticles structures in these cells upon incubation with pre-aggregated gold nanoparticles.

Regardless of the number of internalized aggregates, in both cell culture media and both cell lines, the total number of internalized nanoparticles is higher in the case of incubation with pre-aggregated gold nanoparticles than with non-aggregated nanoparticles.

Therefore, the experiments with pre-aggregated gold nanoparticles serve as a model for higher particle uptake, and differential data can be extracted corresponding to the effects of gold nanoparticles on cells.