Cleavage studies of supercoiled pBR322 DNA by AAPH was performed by agarose gel electrophoresis experiments. DNA damage was measured by the conversion of supercoiled plasmid DNA to open circular and linear forms through single strand nicks and double strand breaks. Radicals for inducing plasmid damage were generated by thermal degradation of AAPH in aqueous solution incubated at 60 ºC for 1 hour. Supercoiled pBR322 plasmid DNA (200 ng) was treated with varying concentrations of AAPH in buffer diluted to a final volume of 10 µL. DNA Samples were vortexed and heated for 1 hr at the indicated temperature to induce DNA damage. After incubation with AAPH, samples were mixed with 2 µL of 6x loading dye and loaded on a 0.8% agarose gel and electrophoresis for 1 hr at 85 V for ~2 hr in 1x Tris-acetate-EDTA (TAE) buffer. For DNA polyplex samples, polplexes were mixed to the desired N/P charge ratio, vortexed and incubated at RT for 10 min. Pre-heated AAPH and 10 mM Tris buffer was added to bring final volume of samples to 10 µL and incubated for 1 hr at the desired temperature. After incubation with AAPH, polyplexes were then decondensed by adding 100 µg of dextran sulfate to each tube and incubated for 10 min at RT before loading samples onto the 0.8% agarose gel. After electrophoresis, gels were stained in ethidium bromide (EtBr, 2.5 ug/mL) for 45 min, then destained in 1x TAE buffer for 30 min. Gel images were captured by UV transillumination with a BioRad ChemiDoc XRS system. Band intensities on gels were quantified using Image Lab 6.0 software (Bio-Rad). To account for the decreased ability of ethidium bromide to intercalate into supercoiled DNA versus nicked DNA and linear DNA, a correction factor of 1.52 was applied to supercoiled DNA. The fraction of each form of DNA in a lane was calculated by dividing the intensity of each band by the total intensities of all bands in the lane.