Abstract
Optical microscopy techniques are ideal for live cell imaging for real-time nanoparticle tracking of nanoparticle localization. However, the quantification of nanoparticle uptake is usually evaluated by analytical methods that require cell isolation. Luminescent labeling of gold nanoparticles with transition metal probes yields particles with attractive photophysical properties, enabling cellular tracking using confocal and time-resolved microscopies. In the current study, gold nanoparticles coated with a red-luminescent ruthenium transition metal complex are used to quantify and track particle uptake and localization. Analysis of the red-luminescence signal from particles is used as a metric of cellular uptake, which correlates to total cellular gold and ruthenium content, independently measured and correlated by inductively coupled plasma mass spectrometry. Tracking of the luminescence signal provides evidence of direct diffusion of the nanoparticles across the cytoplasmic membrane with particles observed in the cytoplasm and mitochondria as nonclustered “free” nanoparticles. Electron microscopy and inhibition studies identified macropinocytosis of clusters of particles into endosomes as the major mechanism of uptake. Nanoparticles were tracked inside GFP-tagged cells by following the red-luminescence signal of the ruthenium complex. Tracking of the particles demonstrates their initial location in early endosomes and, later, in lysosomes and autophagosomes. Colocalization was quantified by calculating the Pearson’s correlation coefficient between red and green luminescence signals and confirmed by electron microscopy. Accumulation of particles in autophagosomes correlated with biochemical evidence of active autophagy, but there was no evidence of detachment of the luminescent label or breakup of the gold core. Instead, accumulation of particles in autophagosomes caused organelle swelling, breakdown of the surrounding membranes, and endosomal release of the nanoparticles into the cytoplasm. The phenomenon of endosomal release has important consequences for the toxicity, cellular targeting, and therapeutic future applications of gold nanoparticles.
Highlights
Gold nanoparticles (AuNPs) have chemical and physical properties that make them unique multimodal probes for medical diagnostics and drug delivery.[1−4] Attractive features include low toxicity and their ability to be functionalized to serve as a “scaffold” for attachment of luminescent probes
The optical signal of the probes is ideal to track the uptake mechanism and the fate of AuNPs inside cells, it has not been utilized as a metric for quantification of AuNPs in cells, which usually relies on the isolation of cells and application of analytical techniques such as inductively coupled mass spectrometry (ICP-MS)
We have shown that analysis of the luminescence signal from particles in confocal images can be used as a metric of cellular uptake and that it is statistically correlated with total cellular gold content quantified by an analytical ICP-MS method
Summary
Gold nanoparticles (AuNPs) have chemical and physical properties that make them unique multimodal probes for medical diagnostics and drug delivery.[1−4] Attractive features include low toxicity and their ability to be functionalized to serve as a “scaffold” for attachment of luminescent probes. Luminescent labeling of AuNPs is a versatile approach for multimodal imaging introducing a luminescent readout signal based on the label while retaining the detection of gold based on its density with either optical or electron microscopy techniques. Luminescent nanoparticles can be monitored in live cells using conventional microscopy techniques, enabling time-resolved information to be collected to track AuNP uptake, fate, and drug delivery.[5] We have previously used luminescent metals to decorate the surface of gold nanoparticles to produce luminescent nanoparticles with a large Stokes shift and high photostability[6−8] and show cellular internalization in the absence of cytotoxicity.[9] We have studied the design of the metal complexes for their distance from the gold particle to eliminate any quenching mechanisms from the gold. The optical signal of the probes is ideal to track the uptake mechanism and the fate of AuNPs inside cells, it has not been utilized as a metric for quantification of AuNPs in cells, which usually relies on the isolation of cells and application of analytical techniques such as inductively coupled mass spectrometry (ICP-MS)
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