Polyethylenimine-mediated impairment of mitochondrial membrane potential, respiration and membrane integrity: Implications for nucleic acid delivery and gene therapy Anna K. Larsen a, ⁎ , 1 , Dominika Malinska b, 1 , Izabela Koszela-Piotrowska b , Ladan Parhamifar a , A. Christy Hunter c , S. Moein Moghimi a, ⁎ a Centre for Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark b Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warszawa, Poland c Molecular Targeting and Polymer Toxicology Group, School of Pharmacy, University of Brighton, Brighton BN2 4GJ, UK abstract article info Article history: Received 21 December 2010 Accepted 31 August 2011 Available online 10 September 2011 Keywords: Apoptosis Mitochondrial membrane potential Mitochondrial swelling Polycations Polyethyleneimine Respiration The 25 kDa branched polyethylenimine (PEI) is a highly efficient synthetic polycation used in transfection protocols, but also triggers mitochondrial-mediated apoptotic cell death processes where the mechanistic is- sues are poorly understood. We now demonstrate that PEI in a concentration- and time-dependent manner can affect functions (membrane potential, swelling and respiration) and ultrastructural integrity of freshly isolated rat liver mitochondria. The threshold concentration for detection of PEI-mediated impairment of rat liver mitochondrial functions is 3 μg/mL, however, lower PEI levels still exert some effects on mitochon- drial morphology and respiration, and these may be related to the inherent membrane perturbing properties of this polycation. The PEI-mediated mitochondrial swelling phase is biphasic, with a fast decaying initial pe- riod (most prominent from 4 μg/mL PEI) followed by a slower, linear swelling response. The slow phase is presumably the result of a time-dependent transition permeability opening in mitochondria initially resistant to swelling/depolarization, but may further be related to PEI-induced nanoscale structural defects and/or for- mation of pores in the outer membrane. Respiration assessments further suggested that PEI in the presence of exogenous ADP behaves in a similar fashion to a slow-acting inhibitory compound. PEI further shows an uncoupling property that is detectable at low respiration rates. The relevance of these findings to PEI- mediated initiation of intrinsic apoptotic pathway is discussed. © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. 1. Introduction Polyethylenimines (PEIs) are a set of commercially available poly- cations with molecular weights ranging from 200 Da to 1500 kDa and are formed in a variety of macromolecular structures (e.g., linear or branched) (Neu et al., 2005; Parhamifar et al., 2010). Every third atom of PEI is a nitrogen capable of undergoing protonation. Accordingly, PEI has been widely used for nucleic acid compaction forming toroidal and globular nanostructures known as polyplexes for experimental gene therapy and silencing (Parhamifar et al., 2010). Polyplexes not only protect nucleic acids from degradation by nucleases, but can favor- ably interact with plasma membranes and efficiently deliver nucleic acids into the cells via different endocytic and macropinocytic mecha- nisms depending on cell type and cell cycle (Hunter and Moghimi, 2010; Parhamifar et al., 2010). PEI exhibits a very high buffering ca- pacity above pH 7, which is attributed to the secondary amines in the structure, but additionally shows buffering capacity in the range of endo-lysosomal pH (Behr, 1997; Kichler et al., 2001). The latter is appar- ently important for intracellular release of nucleic acids, particularly from endosomes. Accordingly, following internalization, PEI may act as a proton sponge buffering the low pH of late endosomes inducing endo- somal rupture; this releases PEI and PEI–nucleic acid complexes into the cytoplasm. The validity of PEI-mediated proton sponge hypothesis, however, has been challenged and alternative escape mechanisms have been proposed (Gabrielson and Pack, 2009; Parhamifar et al., 2010; Rejman et al., 2005; Won et al., 2009). These include polyplex es- cape routes arising from polycation-mediated nanoscale pore formation at the plasma membrane as well as by endosomes (that may result from membrane fragment micellization due to locally induced membrane curvature stress) and/or during fusion processes between cavesomes and early endosomes. The 25 kDa branched PEI is among the most efficient synthetic polycationic transfection reagents, but unfortunately it induces Mitochondrion 12 (2012) 162–168 ⁎ Corresponding authors. E-mail addresses: akl@farma.ku.dk (A.K. Larsen), momo@farma.ku.dk (S.M. Moghimi). 1 AKL and DM contributed equally to this work. 1567-7249/$ – see front matter © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2011.08.013 Contents lists available at SciVerse ScienceDirect Mitochondrion journal homepage: www.elsevier.com/locate/mito