1576 Table II. Mean Diameter (nm) and Zeta Potential (mV) of DNA/p(DMAEA)-ppz Complexes 10 mg DNA/ml, N/P 6 Hepes or HBS Buffer Mean Zeta Potential Hepes Mean Diameter Hepes Mean Diameter HBS Mean Diameter N/P 6 Starting Polymer Ppz950 Ppz570 Ppz290 Ppz130 33 T 6 29 T 2 25 T 5 26 T 7 28 T 1 85 T 4 93 T 7 78 T 1 79 T 3 84 T 6 770 T 250 170 T 12 330 T 13 >1,000 >1,000 175 T 0 226 T 13 182 T 16 N.D. N.D. 150 mg DNA/ml HBG Buffer Mean Diameter N/P 12 162 T 20 195 T 10 128 T 4 152 T 13 145 T 12 de Wolf et al. Mean Diameter N/P 20 N.D. N.D. N.D. 158 T 10 126 T 7 Polyplexes were measured after 1 h of incubation (mean T SD, n = 3). Polydispersity values of the polyplexes were within acceptable limits (<0.3). DNA (Fig. 4). At low ionic strength (Hepes buffer), at N/P ratios of 6 and higher, the extent of DNA binding was similar for the different p(DMAEA)-ppz_s (20% of the original fluorescence remaining). Complexation of DNA with the starting polymer resulted in a considerably stronger DNA binding (residual fluorescence 9–13%). At high ionic strength (HBS), 50% of the original fluorescence was remaining after incubation with the fractionated polymers (data not shown), pointing to a reduced DNA binding capability. Also, the starting polymer showed a lower DNA binding ability, albeit stronger than the fractionated polymers (35% of the original fluorescence remaining). The DNA binding observed for the starting polymer, as reflected by the quenching of EtBr fluorescence, is similar to values observed for linear PEI (Mw 22 kDa, PEI22). Mixing of the different p(DMAEA)-ppz fractions in equal mass ratios yielded less EtBr quenching than observed for the starting polymer. Most likely, a particular molecular weight fraction with a high DNA binding ability was present within the starting polymer and is lost during fractionation. Polyplex Cytotoxicity Cytotoxicity of the different p(DMAEA)-ppz polyplexes was determined using the XTT assay, by comparing the cellular mitochondrial activity of the polyplex treated cells to that of untreated cells. Figure 5 shows the relative cell viability of Neuro 2 A cells, 24 h after transfection with the polyplexes prepared at different N/P ratios. Cytotoxicity positively correlated with polymer molecular weight, in good agreement with earlier findings on PEI, PLL and other polymers (8,12,19,31,32). Polyplexes based on the starting polymer showed significant cytotoxicity, similar to polyplexes based on the high molecular weight polymer fractions (ppz950 and ppz570). Polyplexes based on PEI22 were found to be equally cytotoxic as ppz290 based polyplexes, at the different N/P ratios. At 48 h after transfection, a similar molecular weight correlation was found (data not shown). Recently, the mechanisms underlying the enhanced cytotoxicity of 27 kDa PLL polyplexes was investigated, in comparison to 2.9 kDa PLL polyplexes (12,33). The 27 kDa PLL was found to initiate a stronger, early stage, cell mem- brane perturbation than the low molecular weight polymer, possibly as a result of the more extensive interactions with the cell surface. Moreover, only the 27 kDa PLL and not the 2.9 kDa PLL induced a direct release of cytochrome C from isolated mitochondria, a phenomenon related with late stage cytotoxicity (12). Similar factors might underlie the enhanced cytotoxicity observed for the high molecular weight p(DMAEA)- ppz_s used in this study. Fig. 3. Aggregation of DNA/p(DMAEA)-ppz complexes at high ionic strength. Particle size of complexes based on the starting polymer (circles) and ppz130 (diamonds), after 1 h of incubation in HBS (20 mM Hepes, 150 mM NaCl, pH 7.4) (mean, n = 3). Fig. 4. DNA binding of p(DMAEA)-ppz polymers. DNA binding was measured in Hepes buffer and quantified as percentage residual fluorescence, 1 h after addition of polymer to EtBr labeled DNA (mean T SD, n = 3).