Effect of Polymer Molecular Weight to Tumor Cells 1577 Fig. 5. Cytotoxicity of DNA/p(DMAEA)-ppz complexes towards Neuro 2 A cells, 24 h after incubation (mean T SD, n = 3). IN VITRO TRANSFECTION ACTIVITY The in vitro transfection activity of the different p(DMAEA)-ppz polyplexes was investigated. Figure 6 shows the luciferase expression by Neuro 2 A cells, 24 h after trans- fection with the polyplexes prepared at different N/P ratios. The maximum degree of gene expression was about two orders of magnitude lower than that observed for polyplexes based on PEI22 (N/P 6). Transfection with polyplexes based on the 290 kDa p(DMAEA)-ppz resulted in a bell-shaped curve with highest gene expression levels at N/P ratios of 6 to 50 (Fig. 6d) (19). At low N/P ratios, transfection activity is low, as the polyplexes do not have the favorable physico- chemical characteristics required for efficient cellular adhe- sion, internalization, and a limited extent of (endosomal and nuclear) membrane permeabilization activity (18,19,33). At high N/P ratios, gene expression levels drop most likely due to cytotoxic effects exerted by free, non-DNA associated polymer. In the case of the lowest molecular weight polymer (ppz130), gene expression levels only slightly decrease even at high N/P ratios (Fig. 6e). The low cytotoxicity of the polyplexes based on this polymer enables gene expression even at these high N/P ratios. Transfection with polyplexes based on the starting polymer and the highest molecular weight fraction ppz950 resulted in reduced transfection activity at the intermediate N/P ratios of 6 to 50. Likely, pronounced cytotoxicity at these N/P ratios opposes the transfection process (see polyplex cytotoxicity). Complexes based on ppz570 displayed a somewhat intermediate gene expression profile (Fig. 6c). Polyplexes based on the starting polymer and on ppz950 showed a striking maximum in transfection activity at an N/P ratio of 3 (Fig. 6a and b). To study this phenomenon in more detail, polyplexes based on the starting polymer were prepared in transfection medium with high ionic strength (HBS) and in transfection medium with low ionic strength (HBG). In contrast to the polyplexes prepared in HBS, which rapidly aggregated at N/P ratios of 3 to 6 (diameter 800 to >1000 nm, see also Fig. 3), polyplexes prepared in HBG remained small in time (diameter õ 100 nm at all N/P ratios > 1.5). Transfection activity of the FHBS_ aggregated polyplexes was 15–25 fold higher than their non-aggregated counterparts (Fig. 7). Most likely, complex aggregation in the transfection medium (HBS) is the cause for the observed peak in transfection activity (at N/P 3 to 6, Fig 6a). Earlier, aggregate formation has been found to improve transfection efficiency, likely due to complex sedimentation which enhances the cel- lular contact and subsequent cellular internalization (34,35). Therefore, likely, the polyplex aggregation observed for the dif- Fig. 6. In vitro gene expression of Neuro 2 A cells after transfection with DNA/p(DMAEA)-ppz complexes. Relative light units per well, 24 h after transfection (mean T SD, n = 3).