1578 Fig. 7. Effect of ionic strength on the in vitro transfection activity of DNA/p(DMAEA)-ppz complexes. Transfection values of polyplexes based on the starting polymer, prepared at high ionic strength (HBS) relative to values determined for polyplexes prepared at low ionic strength (HBG), 24 h after transfection of Neuro 2 A cells (mean, n = 3). ferent polymers contributes to the different in vitro gene expression profiles, observed in Fig. 6. Safety Aspects Polyplex-induced erythrocyte aggregation is an impor- tant factor determining the toxicity of polyplexes after intravenous administration (36). The extent of erythrocyte aggregation was studied, 1 h after incubation with the differ- ent polyplexes (150 mg/ml DNA, size 130–230 nm, Table II). At an N/P ratio of 12, the degree of erythrocyte aggregation de Wolf et al. was found to correlate with the molecular weight of the poly- mer used. Extensive aggregation was observed for polyplexes based on the starting polymer and the high molecular weight fractions (ppz950 and ppz570) (Fig. 8a–c). Incubation of the erythrocytes with polyplexes based on the lower molecular weight polymers (ppz290 and ppz130) clearly resulted in a more limited erythrocyte aggregation (Fig. 8d and e). For all polymers, the extent of aggregation was found to be positively correlated with the N/P ratio applied, indicating that free polymer contributes to erythrocyte aggregation (36,37). The extent of polyplex-induced erythrocyte aggregation correlated with the toxicity observed after intravenous administration of the different p(DMAEA)-ppz polyplexes in mice. Tail-vein administration of polyplexes of the starting polymer or the high molecular weight polymers ppz950 and ppz570 was limited to those with N/P ratios of 6 and lower, as lethal toxicity occurred at higher N/P ratios. Remarkably, polyplexes based on the lower molecular weight polymers ppz290 and ppz130 could be administered at two to three fold higher N/P ratios (12 and 20) without causing any apparent toxicity. Polyplex-induced erythrocyte aggregation and sub- sequent capillary occlusion is the likely mechanism linking polymer molecular weight and in vivo toxicity. An associa- tion between polymer molecular weight, erythrocyte aggre- gation and toxicity after intravenous administration has been suggested earlier for PEI polyplexes (38). In Vivo Transfection Tumor and organ gene expression was studied, 24 h after administration of the polyplexes into the tail vein of tumor- bearing mice (N/P ratio of 12 and 20). As a result of occurring toxicity (see safety aspects), gene expression mediated by polyplexes based on the starting polymer and the high Fig. 8. DNA/p(DMAEA)-ppz complex-induced erythrocyte aggregation (representative example at an N/P ratio of 12). Light microscopic image of erythrocytes, after 1 h of incubation with polyplexes based on the starting polymer (a), ppz950 (b), ppz570 (c), ppz290 (d), ppz130 (e).