RESUMO
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.
RESUMO
The development of long-lived luminescent nanoparticles for lifetime imaging is of wide interest as luminescence lifetime is environmentally sensitive detection independent of probe concentration. We report novel iridium-coated gold nanoparticles as probes for multiphoton lifetime imaging with characteristic long luminescent lifetimes based on iridium luminescence in the range of hundreds of nanoseconds and a short signal on the scale of picoseconds based on gold allowing multichannel detection. The tailor-made IrC6 complex forms stable, water-soluble gold nanoparticles (AuNPs) of 13, 25, and 100 nm, bearing 1400, 3200, and 22â¯000 IrC6 complexes per AuNP, respectively. The sensitivity of the iridium signal on the environment of the cell is evidenced with an observed variation of lifetimes. Clusters of iridium nanoparticles show lifetimes from 450 to 590 ns while lifetimes of 660 and 740 ns are an average of different points in the cytoplasm and nucleus. Independent luminescence lifetime studies of the nanoparticles in different media and under aggregation conditions postulate that the unusual long lifetimes observed can be attributed to interaction with proteins rather than nanoparticle aggregation. Total internal reflection fluorescence microscopy (TIRF), confocal microscopy studies and 3D luminescence lifetime stacks confirm the presence of bright, nonaggregated nanoparticles inside the cell. Inductively coupled plasma mass spectrometry (ICPMS) analysis further supports the presence of the nanoparticles in cells. The iridium-coated nanoparticles provide new nanoprobes for lifetime detection with dual channel monitoring. The combination of the sensitivity of the iridium signal to the cell environment together with the nanoscaffold to guide delivery offer opportunities for iridium nanoparticles for targeting and tracking in in vivo models.
