Interaction of Poly(ethylenimine)–DNA Polyplexes with Mitochondria: Implications for a Mechanism of Cytotoxicity

Abstract

Poly(ethylenimine) (PEI) and PEI-based systems have been widely studied for use as nucleic acid delivery vehicles. However, many of these vehicles display high cytotoxicity, rendering them unfit for therapeutic use. By exploring the mechanisms that cause cytotoxicity, and through understanding structure-function relationships between polymers and intracellular interactions, nucleic acid delivery vehicles with precise intracellular properties can be tailored for specific function. Previous research has shown that PEI is able to depolarize mitochondria, but the exact mechanism as to how depolarization is induced remains elusive and therefore is the focus of the current study. Potential mechanisms for mitochondrial depolarization include direct mitochondrial membrane permeabilization by PEI or PEI polyplexes, activation of the mitochondrial permeability transition pore, and interference with mitochondrial membrane proton pumps, specifically Complex I of the electron transport chain and F0F1-ATPase. Herein, confocal microscopy and live cell imaging showed that PEI polyplexes do colocalize to some degree with mitochondria early in transfection, and the degree of colocalization increases over time. Cyclosporine a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporine a was unable to prevent the loss of mitochondrial membrane potential. Further studies done using rotenone and oligomycin to inhibit Complex I of the electron transport chain and F0F1-ATPase, respectively, indicate that both of these mitochondrial proton pumps are functioning during PEI transfection. Overall, we conclude that direct interaction between polyplexes and mitochondria may be the reason why mitochondrial function is impaired during PEI transfection.

Cytotoxicity of polycations: Relationship of molecular weight and the hydrolytic theory of the mechanism of toxicity

“…A common explanation is mitochondrial uncoupling due to pore formation, but evidence relies on isolated mitochondria, and not whole cells (Grandinetti et al, 2011;Larsen et al, 2012). A simpler explanation could be that since polycations damage cell membranes, the cell is simply attempting to maintain homeostatis after sub-lethal damage, by upregulating respiration to provide energy to repair the damage.…”

“…(Parhamifar et al, 2010) It is known that polycationic materials do not produce a apoptotic response, but rather cell death is due to necrosis, (Fischer et al, 2003) and that a variety of organelles are damaged. (Grandinetti et al, 2011;Grandinetti et al, 2012;Moghimi et al, 2005) The mechanism of the necrotic damage is not yet understood. Here we aim to investigate the effect of the properties of the polymer on cell membrane damage.…”

Solid-phase-assisted synthesis of targeting peptide–PEG–oligo(ethane amino)amides for receptor-mediated gene delivery

“…Improved biocompatibility of PEI would be beneficial; PEI of various molecular weights and structures may induce membrane damage when present at higher concentrations, thus causing cell death by apoptosis. 17,18 Also interactions with blood cells 19 and activation of the complement system 20 decrease biocompatibility, efficiency, and target specificity. In this regard, effort has been made to reduce the toxicity of PEI † Electronic supplementary information (ESI) available.…”

Calcium-activated gene transfection from DNA/poly(amic acid-co-imide) complexes

“…Unfortunately, their high positive charge density disrupts cell and mitochondrial membranes, leading to cell necrosis and apoptosis; the inherent cytotoxicity of these cationic polymers, both in vitro and in vivo, continues to be an unresolved problem. 1,2 Equally of concern is the binding of negatively charged serum proteins to positively charged complexes, initiating aggregation and reducing transfection. 3,4 In order to realize the actual clinical potential of these carriers, attempts have been made to modulate the surface charge density by such means as bioconjugation, oxidation, and the addition of anionic materials.…”