title: High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system.
abstract: 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.
authors.affiliation: Centre for Pharmaceutical Nanotechnology and Nanotoxicology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Arnaldur
authors.initials: A
authors.lastname: Hall
authors.forename: Anna K
authors.initials: AK
authors.lastname: Larsen
authors.forename: Ladan
authors.initials: L
authors.lastname: Parhamifar
authors.forename: Kathrine D
authors.initials: KD
authors.lastname: Meyle
authors.forename: Lin-Ping
authors.initials: LP
authors.lastname: Wu
authors.forename: S Moein
authors.initials: SM
authors.lastname: Moghimi
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism* / Q000473:pathology; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D019069:Cell Respiration / Q000187:drug effects*; D004579:Electron Transport / Q000187:drug effects; D045222:Electron Transport Chain Complex Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism; D004734:Energy Metabolism; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008930:Mitochondria, Liver / Q000378:metabolism*; D010084:Oxidation-Reduction; D010085:Oxidative Phosphorylation / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects; D011094:Polyethyleneimine / Q000494:pharmacology*; D011522:Protons
chemical_list: D045222:Electron Transport Chain Complex Proteins; D011522:Protons; D000255:Adenosine Triphosphate; D011094:Polyethyleneimine; D003576:Electron Transport Complex IV
keywords: ((R-L)/E); (L/E); (R/E); Anti-A; Asc; CCCP; CI; CII; CIII; CIV; Cell death; Cytochrome c oxidase; DIC; ETS; Electron transport system; FBS; L; LEAK control ratio; LEAK respiration; MTG; MitoTracker Green; Mitochondrial membrane potential; Mitochondrial uncoupling; Mna; N,N,N′,N′-tetramethyl-p-phenylenediamine; NADH; NaN(3); OXPHOS; PEI; PI; Polyethylenimine; R; RCR; RFRs; ROUTINE control ratio; ROUTINE respiration; ROX; Rote; TMPD; TMRM; antimycin A; ascorbate; carbonyl cyanide m-chlorophenylhydrazone; complex I; complex II; complex III; complex IV; cyt c; cytochrome c; differential interference contrast; electron transport system; fetal bovine serum; malonic acid; netROUTINE control ratio; nicotinamide adenine dinucleotide; oxidative phosphorylation; polyethylenimine; propidium iodide; residual oxygen consumption; respiratory control ratio; respiratory flux ratios; rotenone; sodium azide; tetramethyl rhodamine methyl ester