1 Nanomedicine, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet2 Drug Research Academy A, Drug Research Academy, Faculty of Pharmaceutical Sciences, Københavns Universitet3 Department of Pharmacy, Faculty of Pharmaceutical Sciences, Københavns Universitet4 Section for Metabolic Receptology, The Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, Københavns Universitet5 Drug Research Academy A, Drug Research Academy, Faculty of Pharmaceutical Sciences, Københavns Universitet6 Department of Pharmacy, Faculty of Pharmaceutical Sciences, Københavns Universitet7 Section for Metabolic Receptology, The Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, Københavns Universitet
mitochondrial proton leak and inhibition of the electron transport system
Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in 'broken mitochondria' (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.
Biochimica Et Biophysica Acta, 2013, Vol 1827, Issue 10, p. 1213-1225
Adenosine Triphosphate; Animals; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Membrane; Cell Respiration; Electron Transport; Electron Transport Chain Complex Proteins; Electron Transport Complex IV; Energy Metabolism; Female; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mice; Mitochondria, Liver; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Polyethyleneimine; Protons