# Internalization of Phospholipid-Coated Gold Nanoparticles
## 4 Results
### Endocytosis Occurs in C2C12 and A549 Cells and is Cell Type Specific  
Based on previous studies in our lab, the PCAuNPs were found in acidic compartments [51]. In this work, we sought to clarify which acidic compartments are involved in the uptake of PCAuNPs by studying the extent of their co-localization with markers of compartments associated with the clathrin-mediated endocytic pathway. We incubated cells with PCAuNPs (green) for two hours, followed by fixation and immunofluorescence labeling of either Rab5, Rab7, LAMP-1, and Rab11 compartments in two cell lines, C2C12 and A549 cells. Figure 3 shows contrast enhanced images of C2C12 cells with PCAuNPs (green) and markers for the early endosomes, late endosomes, lysosomes, and recycling endosomes using Rab5, Rab7, LAMP-1 and Rab11, respectively. The images in Figure 3 show that there appears to be little co-localization of PCAuNPs with clathrin-mediated endocytic markers in C2C12 and A549 cells due to the lack of \"yellow\" regions in the images. Quantitative determination of the extent of co-localization using image cross-correlation spectroscopy to obtain the fraction of PCAuNPs that associate with marked compartments is shown in Table 1. It is evident that there is some association of PCAuNPs with each of the four compartments. Figure 3: Endocytic marked compartments containing phospholipid-coated gold nanoparticles in C2C12 and A549 cells. Contrast enhanced fluorescence confocal microscopy images of C2C12 cells and A549 cells with PCAuNPs (green) and Rab5, Rab7, LAMP-1, and Rab11 markers (red) for early, late, lysosomes, and recycling endosomes, respectively. The fractions presented in Table 1 show that 21-33% of the PCAuNPs associate with each marked compartment in C2C12 cells while 38-64% of the PCAuNPs associate with each marked compartment in A549 cells. We can conclude that in both cell lines, these gold nanoparticles are found in the endocytic compartments. It is important to note that, because the markers for the various compartments are not unique [50], some of these compartments will have more than one marker associated with them. This point will be discussed further later. Table 2 shows the fraction of individual marked compartments, or endosomes, that contain PCAuNPs. The fractions presented in Table 2 show 17-20% of each endocytic marked compartment also contains PCAuNPs in C2C12 cells, while only 3-8% of the compartments contain PCAuNPs in A549 cells, with Rab7 compartments being an outlier with about 19% containing PCAuNPs. In previous work, we observed that there was significant co-localization of Rab5 and Rab7 markers, of Rab5 and LAMP-1 markers, and of Rab7 and LAMP-1 markers, demonstrating that these markers are not uniquely defining their respective compartments [50]. We therefore sought to determine the fractions of PCAuNPs that associate with compartments with more than one marker. Accordingly, we incubated cells with phospholipid-coated gold nanoparticles followed by fixation and pairwise immunofluorescence labeling of Rab5 and Rab7; Rab5 and LAMP-1; Rab5 and Rab11; and Rab-7 and LAMP-1. Figure 4 shows contrast enhanced images of C2C12 and A549 cells with PCAuNPs (green) and two pairs of endocytic markers (red and blue) as indicated in the figure. The pink and purple spots in all four images reflect the co-localization of red with blue markers. Consistent with previous work, we observed that there are a few purple and pink spots in all images to indicate co-localization of Rab5 with Rab7, Rab11 and LAMP-1, and of Rab7 with LAMP-1 [50]. The association of PCAuNPs and Rab5 would be shown by a yellow color and the association of PCAuNPs with Rab7 and LAMP-1 would be shown by a cyan. Neither yellow nor cyan are present to a high extent. Co-localization of PCAuNPs with two markers would be observed as white regions.