# Internalization of Phospholipid-Coated Gold Nanoparticles
## 5 Discussion  
Our objective was to address three questions about the uptake of phospholipid-coated gold nanoparticles: (1) Are they internalized by clathrin-mediated endocytosis; (2) Does the process depend on cell type, and (3) What happens to them with time? The images in Figure 2 show that the PCAuNPs are internal to the cells and that they co-localize with intracellular compartments. The cross-correlation data in Table 1 unambiguously show that these PCAuNPs are internalized in both cell lines into compartments that also contain markers that have been identified with early, late, and recycling endosomes as well as lysosomes. This is consistent with the previous observation that they are found in acidic compartments [51] in A549 cells. It is \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Time (Hours)** & \(\langle\)\(N_{AuPCNP}\rangle\) & \(\langle\)\(CD\)\(\rangle\) & \(\langle\)\(DA\)\(\rangle\) \\ \hline
1 & \(11.4\pm 2.1\)\({}^{+}\) & \(21.4\pm 3.6\) & \(16.4\pm 4.6\) \\ \hline
2 & \(6.96\pm 1.33\) & \(14.8\pm 2.5\) & \(40.2\pm 15.8\) \\ \hline
4 & \(3.73\pm 0.62\) & \(8.91\pm 1.70\) & \(71.2\pm 15.8\) \\ \hline
24 & \(0.93\pm 0.16\) & \(1.49\pm 0.38\) & \(700\pm 49\) \\ \hline \hline \end{tabular} \({}^{+}\)Standard error.  
\end{table} 
Table 6: Averages of derived parameters for PCAuNPs undergoing variable exposure with fixed uptake in A549 cells. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Time (Hours)** & \(\langle\)\(g(0,0)\)\(\rangle\) & \(\langle\)\(\omega\)\(\omega\)\(\mu\)m\(\rangle\) & \(\langle\)\(I_{\text{ang.}}\rangle\) \\ \hline
1 & \(0.21\pm 0.04\)\({}^{+}\) & \(0.41\pm 0.01\) & \(82.8\pm 8.3\) \\ \hline
2 & \(0.27\pm 0.07\) & \(0.39\pm 0.02\) & \(131\pm 15\) \\ \hline
4 & \(0.65\pm 0.17\) & \(0.38\pm 0.01\) & \(138\pm 10\) \\ \hline
24 & \(1.61\pm 0.18\) & \(0.48\pm 0.01\) & \(527\pm 43\) \\ \hline \hline \end{tabular} \({}^{+}\)Standard error.  
\end{table} 
Table 5: Averages of ICS Parameters for PCAuNPs after variable exposure with fixed uptake in A549 cells. also clear from the data in Tables 1 and 2 that the gold nanoparticles are associated with one of these four compartments, but that only a small fraction of the endosome compartments contain these gold nanoparticles. The triple cross correlation data in Tables 3 and 4 confirm the presence of the gold nanoparticles in compartments with multiple markers, which is consistent with recent observations that these markers are not unique to particular endosomes [50]. Thus, we conclude that these PCAuNPs internalize via the clathrin-mediated endocytosis pathway in both cell lines. Both the auto- and cross-correlation data suggest that even though the extent of uptake of gold nanoparticles is similar in C2C12 and A549 cells (Figure 6), their distribution within the cells differ. In the A549 cells, the PCAuNPs appear in a smaller fraction of the endosome compartments than in C2C12 cells (Tables 2 and 4), which is consistent with the cluster density in A549 cells being lower than in the C2C12 cells (Figure 6). Thus, there are more PCAuNPs present in fewer compartments and they are therefore in a more aggregated state in A549 cells. It is not clear why this difference exists, but A549 cells are derived from type II alveolar cells that are designed to recycle liposomes of dimensions comparable to these gold nanoparticles [64]. As they are coated with phospholipids, the cell may be processing them as if they are liposomes [65], perhaps into lamellar bodies. After a short exposure to the gold nanoparticles, the A549 cells appear to dispose of them over a 24-hour period (Figure 6 and Table 5). The number of clusters decrease as does the extent of aggregation within them. This is consistent with the observation from the cross-correlation experiments (Tables 1 and 2) that some of the PCAuNPs associate with Rab11, which is used as a marker for the recycling endosomes. They may offer a mechanism whereby the PCAuNPs are expelled from the cells.