Low and high molecular weight poly(l‐lysine)s/poly(l‐lysine)–DNA complexes initiate mitochondrial‐mediated apoptosis differently

Poly(l‐lysine)s, PLLs, are commonly used for DNA compaction and cell transfection. We report that, although PLLs of low (2.9 kDa), L‐PLL, and high (27.4 kDa), H‐PLL, Mw in free form and DNA‐complexed cannot only cause rapid plasma membrane damage in human cell lines, phosphatidylserine “scrambling” and loss of membrane integrity, but later (24 h) initiate stress‐induced cell death via mitochondrial permeabilization without the involvement of processed caspase‐2. Mitochondrially mediated apoptosis was confirmed by detection of cytochrome c (Cyt c) release, activation of caspases‐9 and ‐3, and subsequent changes in mitochondrial membrane potential. Plasma membrane damage and apoptosis were most prominent with H‐PLL. Cytoplasmic level of Cyt c was more elevated following H‐PLL treatment, but unlike L‐PLL case, inhibition of Bax channel‐forming activity reduced the extent of Cyt c release from mitochondria by half. Inhibition of Bax channel‐forming activity had no modulatory effect on L‐PLL‐mediated Cyt c release. Further, functional studies of isolated mitochondria indicate that H‐PLL, but not L‐PLL, can directly induce Cyt c release, membrane depolarization, and a progressive decline in the rate of uncoupled respiration. Combined, our data suggest that H‐PLL and L‐PLL are capable of initiating mitochondrially mediated apoptosis differently. The observed PLL‐mediated late‐phase apoptosis may provide an explanation for previously reported transient gene expression associated with PLL‐based transfection vectors. The importance of our data in relation to design of novel and safer cationic non‐viral vectors for human gene therapy is discussed.