1574 trations (1–10 mg/ml) directly into the refractive index detector. Molecular weight data analysis was performed by OmniSEC 4.1 software. An 31P-Nuclear magnetic resonance (NMR) spectrum of the fractionated polymers was recorded on a Varian Inova 500 MHz spectrometer (Varian, USA) (22). Preparation of the Polyplexes Plasmid DNA encoding for firefly luciferase (pcDNA3- Luc) was used for all experiments. The molar polymer-to-DNA (N/P) ratio of the polyplexes varied from 1.5 to 100. A mass per charge of 110 and 330 Da was used for p(DMAEA)-ppz and DNA, respectively. Polyplexes for the in vitro experiments were prepared at a DNA concentration of 10 mg/ml. The buffers used for the preparation of the polyplexes had either a low ionic strength (5 mM Hepes, pH 7.4 or 20 mM Hepes, 5% g/v glucose, pH 7.4, HBG) or a high ionic strength (20 mM Hepes, 150 mM NaCl, pH 7.4, HBS). Polyplexes were prepared by adding four volumes of the polymer solution to one volume of the DNA solution. The resulting dispersions were vortexed and incubated for 30 min at room temperature before use (26). Polyplexes for the in vivo studies, and for the in vitro erythrocyte aggregation assays were prepared at a DNA concentration of 150 mg/ml (20 mM Hepes, 5% g/v glucose, pH 7.1) by adding one volume of a polymer solution to one volume of DNA solution. The resulting dispersions were vortexed and incubated for 30 min at room temperature. Then, one tenth a volume of a 50% g/v D-glucose stock was added to obtain iso-osmolarity (27). Characterization of the Polyplexes The average hydrodynamic diameter and the zeta- potential of the polyplexes were determined using dynamic light scattering and electrophoretic mobility measurements, respectively, at a DNA concentration of 10 mg/ml. The size of the polyplexes was determined using an ALV CGS-3 system (Malvern Instruments, UK). The zeta-potential of the poly- plexes was determined using a Zetasizer 2000 (Malvern instruments, UK). Both instruments were calibrated using polystyrene latex beads of defined size and zeta-potential. Condensation of DNA by the polymers was determined using an ethidium bromide (EtBr) fluorescence quenching assay (28). EtBr was added to a DNA solution 10 min prior to polyplex formation at a molar ratio of 1:8. Next, EtBr labeled polyplexes were prepared at a DNA concentration of 10 mg/ml in Hepes or HBS, as described earlier. The resulting fluorescence was quantified using a Fluorstar Optima plate- reader (BMG Lab Tech, Germany). Fluorescence values were measured at an excitation/emission wavelength of 500 and 600 nm, respectively, and converted into relative, residual fluorescence (Fr) using the following equation: Fr ¼ ðFobs FeÞ=ðF0 FeÞ; where Fobs is the observed fluorescence of the polyplex samples, Fe is the fluorescence of EtBr in the absence of DNA, and F0 is the fluorescence of EtBr in the presence of DNA. de Wolf et al. In Vitro Transfection Neuro 2 A (Murine Neuroblastoma) cells (ATCC CCL- 131) were cultured in RPMI 1640 medium completed with bovine calf serum (10%) and antibiotics/antimycotics (100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin B) at 37-C at a 5% CO2 humidified atmosphere. Transfection and cytotoxicity studies were carried out in serum containing medium, as described earlier (5). In brief, cells were seeded in a 96-well plate at a density of 1 104 cells/well. After twenty-four hours, cells were overlaid with fresh medium (100 ml) and subsequently overlaid with a polyplex dispersion (100 ml, 1 mg DNA, HBS or HBG). Cells were incubated with the polyplexes for 1 h. Subsequently, the medium was refreshed and the cells were cultured for another 24 h. All transfection experiments were carried out in duplicate in two separate 96-well plates. One series was tested for reporter gene expression (luciferase activity) (5), the other series was tested for cell viability using an XTT colorimetric assay (29). Luciferase activity was measured after lysis of the cell pellet by the addition of 100 ml reporter gene lysis buffer fol- lowed by a single freeze/thaw cycle. Luciferase activity of the samples was determined using a Berthold 9507 Luminometer (EG&G Benelux BV, The Netherlands) by mixing 20 ml of the defrosted cell lysate with 100 ml of luciferase assay reagent. Relative light units (RLU) were measured for 10 s at room temperature and were corrected for background values. In this setting, 6 103 RLUs corresponded to 1 pg of recombinant luciferase. The percentage of viable cells was tested using an XTT colorimetric assay, based on the metabolic, cellular reduction of a tetrazolium reagent (29). Cell viability values were nor- malized to values obtained after incubation with buffer only. Polyplex-induced Erythrocyte Aggregation Mouse erythrocytes were isolated from heparinized blood by centrifugation. The erythrocyte pellet was washed several times with HBS buffer. Subsequently, a suspension was prepared by diluting the erythrocyte pellet 40 times with HBS. To 20 ml of the different polyplex dispersions (150 mg/ml DNA in 20 mM Hepes, 5% g/v glucose, pH 7.1, N/P ratio 12), 230 ml of the erythrocyte suspension was added. After 1 h of incubation at 37-C, aliquots of the suspensions were taken for microscopic analysis (20 ). In Vivo Gene Expression Studies The animal experiments were performed according to national regulations and approved by the local animal experiments ethical committee. Six-week old male A/J mice (Harlan, The Netherlands) were inoculated with 1 106 Neuro 2 A cells in the left flank. Eight to twelve days after inoculation, at an average tumor volume of 0.4–0.8 cm3, the different polyplex dispersions were injected into the tail-vein of the mice (0.2 ml, corresponding with 30 mg DNA). The mice were sacrificed 24 h after injection by cervical dis- location and luciferase levels of the different organs were assessed as previously described (30). Briefly, organs were homogenized in 0.5–1 ml of reporter gene lysis buffer, using a tissue homogenizer. The tissue homogenates were incubated