NOVEL APPLICATION OF FLUORESCENT SEMICONDUCTOR NANOCRYSTALS. TARGETED DELIVERY OF QUANTUM DOTS DURING HYPOTHERMIC PRESERVATION OF HEPATOCYTES

VALERIA SIGOT; JOAQUÍN RODRÍGUEZ; JOSÉ PELLEGRINO; EDGARDO GUIBERT

Gramado

Congreso; VI Congreso Latinoamericano de Órganos Artificiales y Biomateriales-COLAOB2010; 2010

Sociedad Latinoamericana de Biomateriales

The delivery of biological and /or inorganic cargo across the membrane lipidbilayer of living cells towards the cell interior has many potential applications, ranging from the development of new therapeutics agents to the basic understanding of membrane transport mechanisms. Quantum Dots (QDs) provide a more photo-stable, brilliant and less toxic alternative to conventional organic fluorophores for long term fluorescent imaging. We evaluate the novel application of QDs as nanoprobes to target cultured rat hepatocytes preserved in University of Wisconsin solution at 4°C up to 72 h. Previous work showed that QDs conjugated to Epidermal Growth Factor (EGF) ligand internalize by receptor-mediatedendocytosis at 37°C. We aim to monitor the uptake of EGF-QDs complexes in primary cultures of rat hepatocytes (CH) and in cultured and cold-stored rat hepatocytes (CCSH) to evaluate the effect of hypothermic preservation on membrane integrity and vesicular transport. Biotinylated-EGF was conjugated to streptavidin-coated Quantum Dots (QDs) with fluorescence emission at 655 nm or 585 nm. QDs fluorescence was detected down to picomolar concentrations. We monitored the fate of EGF-QDs complexes by confocal microscopy in CH incubated with EGF-QDs complexes 1 h at 37°C in culture medium and in CCSH incubated with EGF-QDs in UW up to 72 h at 4°C. We observed specific internalization of the complexes in CH at 37°C, whereas no binding was detected after incubation with QDs (without EGF). During cold storage in UW at 4°C, where EGF stimulation is inhibited, EGFQDscomplexes remained bound and concentrated on the cell membrane of CCSH. Currently, we evaluate the uptake of EGF-QDs in CCSH after rewarming at 37°C. Potential application will be the loading of targeted Quantum Dots with a therapeutic biomolecule that can concentrate on the cell membrane during preservation and can exert its effect after returning to normothermia.