Lymphocyte death by cationic polymers: A role for mitochondrion and implications in gene therapy

A wide range of synthetic polycations in linear, branched, or dendrimer form have been used to condense DNA into structures amenable to cellular internalization via endocytosis. Polycations can destabilize endosomal membranes or act as proton sponges; they buffer the low pH in the endosomes and potentially induce membrane rupture, resulting in the release of polycation/DNA complex into the cytoplasm. The polycationic nature of the gene-delivery vehicles can induce cytotoxicity, but the mechanisms are poorly understood. We studied the effect of a number of commonly used polycations on mitochondrial functions in isolated mitochondria from rat liver as well as directly in Jurkat cells. Upon permeabilization or rupture of the outer mitochondrial membrane, cytochrome c binds to Apaf-1, leading to allosteric activation of pro-caspase-9. This in turn proteolytically activates caspase-3, one of the principal proteases that participates in the execution of cell death. A decrease in mitochondrial membrane potential (∆ϕ) due to permeability transition is also an early event in several types of apoptosis. We have demonstrated that at very low concentrations, polycations can affect mitochondrial respiration and ∆ϕ; these events were followed by cytochrome c release from mitochondrial intermembrane in mitochondrial suspensions and in Jurkat cells. Changes in mitochondrial∆ϕ in Jurkat cells were confi rmed by the Mitosensor test. Detection of phosphatidylserine translocation to the cell surface and proteolytic activation of caspase-3 by apoptosome further confi rmed activation of apoptosis in Jurkat cells. These observations provide a molecular explanation for previously reported immediate or delayed cytotoxicity following gene transfer with polycations. The results from this study may help to design novel materials with high transfection effi ciencies suitable for clinical gene therapy