? Major limitations in liver transplantation are primary graft failure leading to consumption of the already small donor pool. ATP depletion has been proposed to be involved in graft failure due to both prolonged storage and to transplantation of marginal fatty livers: Our recent developments of a fusogenic lipid vesicles that deliver ATP are therefore a promising therapy to treat primary nonfunction. The goals of the proposed work are to characterize and optimize existing formulations of our lipid vesicles for delivery of ATP to protect against primary graft failure. First, the rate of fusion of lipid vesicles to liver cells and delivery of ATP will be determined using cultured primary parenchymal and non-parenchymal (endothelial and Kupffer) cells. We will then determine the effect of delivery of ATP on cell death caused by hypoxia in culture. From these experiments, the 3 most promising formulations will be further studied. Next, livers will be cold-stored in UW solution (0-48 h) and subsequently perfused on a liver perfusion block as a model of cold storage -induced reperfusion injury; UW solution containing our vesicles (0-5 mg/mL) and Mg-ATP (0-10 mM) will be infused into the organ at the initiation of storage (0-48 h). To mimic primary graft failure due to acute ethanol administration, some animals will be pretreated with ethanol (5 g/kg i.g.) 20 h prior to explant surgery. Primary endpoints include cell death, the production of free radicals, and indices of metabolic activity and redox states. From these experiments, the most effective vesicle preparation will be identified. Taken together, the expected results of this proposed work will identify optimal conditions for delivery of ATP to cold-stored livers with lipid vesicles. Such results would not only serve as proof-of-principle of the long-standing hypothesis that ATP pools are critical during liver transplant, but also identify a unique therapy that could then be readily applied to the clinical situation, where such a therapy is critically needed. ? ? ?
