Effect of hypothermic preservation on targeted delivery of EGF-Quantum Dots complexes to primary culture of rat hepatocytes

HOVANYECZ, P.; RODRIGUEZ, J. V.; GUIBERT, E. E.; PELLEGRINO, J. M.,; BOTTAI, H.; SIGOT, V

Encuentro; 49th Annual Meeting of the Society for Cryobiology; 2012

Centro Binacional (Argentina-Italia) de Investigaciones en Criobiología Clínica y Aplicada

During cell and tissue hypothermic preservation (HP) at 4°C, metabolic rate is diminished and energy-dependent processes such as endocytosis are arrested. The onset of cold induced injury is expressed after rewarming to normothermia, which in turns accelerates loss of membrane integrity and cell death. Receptor-mediated endocytosis is an energy dependent mechanism requiring intact membranes as well as the integrity of the cytoskeleton for efficient activation and internalization of ligand-receptor complexes. The Epidermal Growth Factor Receptor (EGFR) endocytic pathway is temperature sensitive. However, studies under hypothermia were carried out for short periods (1 h at 4°C), during which membrane integrity was not compromised. We evaluated the effect of hypothermic preservation on EGFR mediated endocytosis in rat hepatocytes by monitoring the uptake of fluorescent Quantum Dots (QDs) conjugated to the EGF ligand after normothermic rewarming (NR). The EGF-QDs complexes were added to cultured rat hepatocytes (CH) and to cold-stored hepatocytes (CSH) preserved in University of Wisconsin solution (UW) at 4°C for 24 h (CSH24) and 72 h (CSH72).  After HP hepatocytes were incubated with preformed complexes of EGF-QDs (at 8:2 molar ratio) followed by 30 and 120 min incubation at 37ºC in culture medium under 5% CO2 atmosphere (NR) and the uptake of QDs was visualized by fluorescence confocal microscopy. Membrane integrity, and thus cell viability, was measured based on the release of the enzyme Lactate Dehydrogenase (LDH). At each time point of HP and NR the extracellular activity of LDH was determined in culture medium or in UW solution. Results were expressed as the percentage of total enzyme activity released in the extracellular medium. Cytoskeleton distribution after HP and NR was visualized by fluorescence microscopy after actin staining with Phalloidin-Alexa-633. Confocal microscopy showed targeted QDs internalized after 30 min NR in CSH24 but not in CSH72, while maintaining a normal cytoskeleton distribution. Comparable amounts of LDH were released by CSH24 and CSH72 in UW solution (0 min NR) indicating that viability is not altered during the cold storage. However, in CSH72 after 30 min NR, LDH released increased form 15% to 30% These results showed that after 24 hours of cold storage and 30 min NR hepatocytes are competent for receptor mediated uptake but extended preservation (up to 72 h) mainly affects the ability to internalize but no bind the EGF-QDs. Internalization of non-targeted QDs was not observed, indicating that unspecific binding or passive diffusion did not occur. These observations suggest that loss in membrane integrity may affect the endocytic uptake mediated by EGFR after 72 h HP followed by 30 min NR. Additional experiments to characterize the energy status (ATP levels) will be addressed. Targeted QDs may act as suitable fluorescent biosensors for cold-induced membrane injury and its effect on the EGF-receptor mediated transport at the level of individual cells, complementing the LDH release global test for membrane integrity.