Copper is a trace metal found in all living organisms, both in the oxidized Cu2+ and reduced Cu+ states. In physiological conditions, copper homeostasis is a complex mechanism, which involves its uptake, efflux and compartmentalization within sub-cellular districts, and relies on dynamic association/dissociation processes among a certain number of transporters, chaperones and small-molecule ligands. Temporal and spatial changes in intracellular copper distribution have been demonstrated to play a key role both at the onset and in the advancing stages of several diseases, including neurodegenerative disorders and cancer, but the precise copper role in inducing pathological changes remains poorly understood. We present here a novel nanoparticle-based strategy to image intracellular copper distribution and to follow its dynamic changes upon cell ‘perturbation’. Porous core-shell silica nanoparticles, having average dimensions of 25 nm and 10 nm respectively for external and internal diameters, were synthesized and dye-doped both in the core (coumarin fluorophore) as well as on the outer shell (bodipy fluorophore). In particular, the bodipy units was covalently linked to a highly specific and selective Cu+ chemosensor. Live cell imaging by confocal microscopy was used to scrutinize the localized cell lighting upon cell perturbation induced by the addition of exogenous copper, the interaction and internalization of peptide fragments, and pH changes. The results evidence the superior imaging capability of the nanoparticle-based sensor compared with the free Cu+ chemosensor, due to both the energy-transfer process from coumarin to bodipy fluorophore as well as the higher Cu+ binding affinity . The present approach is very promising for real time detection of dynamics of labile copper pools in the intracellular environment and thus the understanding of sub-cellular biochemical pathways responsible for the disease onset and progression. Acknowledgements: The authors thank the financial support from MIUR (FIRB MERIT - RBNE08HWLZ and FIRB ITALNANONET – RBPR05JE2P).
Nanoparticle-Enhanced Fluorescence Microscopy As New Tool For Monitoring Intracellular Copper Trafficking
SATRIANO, Cristina;COPANI, Agata Graziella;TOMASELLI, Gaetano;
2012-01-01
Abstract
Copper is a trace metal found in all living organisms, both in the oxidized Cu2+ and reduced Cu+ states. In physiological conditions, copper homeostasis is a complex mechanism, which involves its uptake, efflux and compartmentalization within sub-cellular districts, and relies on dynamic association/dissociation processes among a certain number of transporters, chaperones and small-molecule ligands. Temporal and spatial changes in intracellular copper distribution have been demonstrated to play a key role both at the onset and in the advancing stages of several diseases, including neurodegenerative disorders and cancer, but the precise copper role in inducing pathological changes remains poorly understood. We present here a novel nanoparticle-based strategy to image intracellular copper distribution and to follow its dynamic changes upon cell ‘perturbation’. Porous core-shell silica nanoparticles, having average dimensions of 25 nm and 10 nm respectively for external and internal diameters, were synthesized and dye-doped both in the core (coumarin fluorophore) as well as on the outer shell (bodipy fluorophore). In particular, the bodipy units was covalently linked to a highly specific and selective Cu+ chemosensor. Live cell imaging by confocal microscopy was used to scrutinize the localized cell lighting upon cell perturbation induced by the addition of exogenous copper, the interaction and internalization of peptide fragments, and pH changes. The results evidence the superior imaging capability of the nanoparticle-based sensor compared with the free Cu+ chemosensor, due to both the energy-transfer process from coumarin to bodipy fluorophore as well as the higher Cu+ binding affinity . The present approach is very promising for real time detection of dynamics of labile copper pools in the intracellular environment and thus the understanding of sub-cellular biochemical pathways responsible for the disease onset and progression. Acknowledgements: The authors thank the financial support from MIUR (FIRB MERIT - RBNE08HWLZ and FIRB ITALNANONET – RBPR05JE2P).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.