title: 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 F(0)F(1)-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. Cyclosporin a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporin 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 F(0)F(1)-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.
authors.affiliation: Department of Chemistry & Macromolecules & Interfaces Institute, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Grandinetti
authors.forename: Nilesh P
authors.initials: NP
authors.lastname: Ingle
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Reineke
mesh_terms: D017209:Apoptosis; D001692:Biological Transport / Q000187:drug effects; D053453:Caspase 9 / Q000378:metabolism; D002470:Cell Survival; D058948:Cell Tracking; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism*; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry; D018014:Gene Transfer Techniques / Q000009:adverse effects*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008422:Materials Testing; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010539:Permeability; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011094:Polyethyleneimine / Q000009:adverse effects* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004270:DNA, Circular; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D011094:Polyethyleneimine; C506433:CASP9 protein, human; D053453:Caspase 9; D025261:Mitochondrial Proton-Translocating ATPases; D042967:Electron Transport Complex I