Uptake and intracellular localization of engineered gold nanoparticles in A549 cells

Engineered gold nanoparticles (AuNPs) possess unique physiochemical properties such as large surface area to mass ratio. Their relative ease of synthesis including incorporation of transition metal-ligands and other labelling... [ view full abstract ]

Engineered gold nanoparticles (AuNPs) possess unique physiochemical properties such as large surface area to mass ratio. Their relative ease of synthesis including incorporation of transition metal-ligands and other labelling agents produces functionalized AuNPs with potential applications for therapeutic and diagnostic medicine. Prior to clinical applications, an understanding of uptake, localization and cellular fate in relation to surface properties is critical.

In the current study we synthesised 13 nM AuNPs coated with a luminescent ruthenium metal complex to give rise to RubpySS.AuNP13. A non-toxic concentration (0.9 nM, MTT assay) was chosen to study particle uptake in A549 human lung carcinoma cells. Cells were incubated with RubpySS.AuNP13 (2-72 hours), uptake and co-localisation with different cellular compartments was studied using fluorescent probes (mitochondria, endoplasmic reticulum and golgi) and GFP-tagged organelle markers (early endosome, lysosome and autophagosome) by confocal microscopy supplemented by transmission electron microscopy (TEM). The size of particles in cells as quantified by TEM was in agreement with characterisation data of particles in solution (DLS and TEM) and particle size remained unchanged after up to 72 hours incubation indicating that RubpySS.AuNP13 does not aggregate upon cellular uptake. Particle number increased over time and this was confirmed by quantitative analysis (ImageJ) and further supported by TEM.

Both confocal and TEM images suggested the involvement of the endo-lysosomal pathway as a route of cellular uptake of RubySS.AuNP13 in A549 cells. RubySS.AuNP13 showed no significant co-localisation with markers for mitochondria, endoplasmic reticulum and the golgi and this was confirmed by TEM. In contrast, we observed particles in the early endosomes at early time points (4 hours), after 12-72 hours particles were observed to accumulate in a time-dependent manner in both the lysosomal and autophagosomal compartments of the cell, again this was confirmed by TEM. Also evident was a change in diffuse cytoplasmic LC3 staining to more punctuate staining consistent with the formation of active autophagosomes. In conclusion, we report that a major route of cellular uptake of RubySS.AuNP13 is by the endo-lysosomal pathway and furthermore accumulation of particles into autophagosome may have important implications for their cellular fate and toxicity.