Contaminants Within Bacterial Plasmid Preparations Trigger Apoptosis in Liposome Transfected Ovcar3, but Not in Skov3 or Az224 Human Ovarian Cancer Cells

Abstract

Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.

High-throughput isolation of ultra-pure plasmid DNA by a robotic system

“…Figure 2B reveals that the DNAs from the commercial columns were less efficiently transfected and also adversely affected the cells as evident by their detachment and rounding up (arrows). The latter observation is of crucial importance, since, while transfections with less well purified plasmid DNA could potentially be obtained in some cells, the effects of the plasmid DNA can only be observed if the cells are not damaged by residual bacterial components[ 20 ]. We concluded that our robotic plasmid preparations are superior both in costs and quality compared to the currently available ways to obtain DNA in a 96-well format.…”

Lipid Nanoparticle Formulation Increases Efficiency of DNA-Vectored Vaccines/Immunoprophylaxis in Animals Including Transchromosomic Bovines

“…It is also possible that the combination of DNA and the LNP triggers an "off-target" response similar to cationic lipoplex [27][28][29] . Alternatively, contaminants from DNA production (bacterial derived) may also stimulate cellular responses 30,31 .…”