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

During cell and tissue hypothermic preservation (HP) at 4 °C, metabolism decelerates and energy-dependent processes such as endocytosis are arrested. Cold induced injury expressed after rewarming to normothermia, accelerates loss of membrane integrity and cell death. Receptor-mediated endocytosis is a temperature and energy dependent mechanism requiring intact membranes as well as the integrity of the cytoskeleton for efficient activation and internalization of ligand–receptor complexes. We evaluated the effect of hypothermic preservation on epidermal growth factor (EGF) receptor mediated endocytosis in cultured and cold preserved rat hepatocytes after normothermic rewarming (NR). Fluorescent streptavidin-Quantum Dots (QDs) emitting at 655 nm were conjugated to biotin-EGF at 8:2 molar ratio. 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 10 min at 10 °C to promote binding but not internalization, followed by 30 and 120 min incubation at 37 °C in culture medium under 5% CO 2 atmosphere (NR) and the uptake of QDs was visualized by fluorescence confocal microscopy. Non-targeted QDs were added under the same incubation conditions as controls for unspecific binding. At each time point of HP and after 30 and 120 min NR, membrane integrity, and thus cell viability, was determined based on the activity of the enzyme lactate dehydrogenase (LDH), expressed as the percentage of total enzyme retained in the hepatocytes. 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 CH and in CSH24 but not in CSH72 which only showed QDs concentrated on the membranes. Internalization of non-targeted QDs was not observed, indicating that unspecific binding was not occurring. After 30 min NR, cytoskeleton distribution in CSH24 and in CSH72 was comparable to that of HC. LDH retention was in, HC: 90.1 ± 2.1%, CSH24: 89.8 ± 1.7% and in CSH72:72.5 ± 5.8% ( p p These results showed that after 24 h of cold storage and 30 min NR, rat 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 while cells are still viable. ATP depletion associated with extended cold preservation may account for the lack of activation of EGFR and internalization of QDs observed after NR. Therefore, additional experiments to assess energy status of hepatocytes after cold storage may contribute to the understanding of the energy dependence of this receptor mediated transport. Targeted QDs may act as suitable fluorescent biosensors for cold-impaired endocytic transport after normothermic rewarming giving insight into cell membrane damage at the level of specific surface receptors.