Table I. Characterization of the Starting Polymer and the Obtained Polymer Fractions Fraction Starting ppz Ppz950 Ppz570 Ppz290 Ppz130 Mw (kDa) Mn (kDa) Mw/Mn Rh (nm) 680 950 570 290 130 390 850 440 250 100 1.8 1.1 1.3 1.2 1.3 Effect of Polymer Molecular Weight to Tumor Cells on ice for 30 min, then vortexed and subsequently centrifuged at 12,000 g for 10 min. The luciferase activity of the samples was determined using a Berthold 9507 Luminometer by mixing 20 ml of the supernatant 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 re- combinant luciferase. The transfection activity of the poly- plexes was expressed as RLU/organ or RLU/tumor. RESULTS AND DISCUSSION Polymer Synthesis A batch of polymer p(DMAEA)-ppz was synthesized and purified as previously described (22). The SEC chromatogram of this starting polymer is shown in Fig. 2a. The polymer had a weight average molecular weight of 680 kDa (Table I). The measured molecular weight distribution was around 2 and is a consequence of the method of polymerization (22–24). Fractionation Aqueous size exclusion chromatography (SEC) was used for fractionation and analysis of the p(DMAEA)-ppz poly- mers. The combined, online use of a series of detectors (low Fig. 2. SEC chromatogram of the starting polymer (a) and polymer fraction ppz290 (b). Left axis: low angle light scattering (bright grey), viscosity (medium grey), refractive index (dark grey). Right axis: calculated molar mass dependency (black). 1575 21 26 19 13 8 and right angle light scattering, viscosity, refractive index) enabled the determination of the absolute molecular weight of the fractionated polymers, as well as their molecular weight distribution. The refractive index increment (dn/dc) of p(DMAEA)-ppz was determined to be 0.159 ml/g. Figure 2b shows the SEC chromatogram of one of the fractions of p(DMAEA)-ppz (ppz290). The molecular weight distribution of this fractionated polymer was relatively narrow (Mw/Mn 1.2, Table I). The same held true for the other fractionated polymers (Mw/Mn 1.1–1.3). The weight average molecular weight of the different polymers ranged from 130 to 950 kDa which corresponds to hydrodynamic radii ranging from 8 to 26 nm. The dependency of the calculated molecular weight on retention volume was found to be linear for the different polymers, demonstrating good SEC separa- tion conditions. 31P-NMR analysis of the selected fractions showed one peak at 1.7 ppm (22) indicating that no polymer degradation had occurred during the fractionation process. Biophysical Characterization Polyplexes with a mean size between 80 to 90 nm were formed upon complexation of the different polymer fractions with plasmid DNA at a relatively low DNA concentration (10 mg/ml) and at low ionic strength (Hepes buffer), independent of the polymer molecular weight (Table II). All polyplexes had a positive surface charge (zeta-potential 25–29 mV). At high ionic strength (HBS), the different polyplexes gradually aggregated. Most likely, aggregation occurs due to the reduction in zeta-potential of the particles which leads to a decrease in interparticle repulsion and colloidal stability. Aggregate formation was observed over a broad range of N/P ratios in the case of the polyplexes based on the smaller molecular weight polymers (ppz290 and ppz130), whereas the higher molecular weight polymers showed aggregate formation only at N/P ratios of 3 to 6 (starting polymer, ppz950 and ppz570, Fig. 3). The low col- loidal stability of the polyplexes based on the low molecular weight polymers is in good agreement with earlier findings on PEI, pLL, and pDMAEMA polyplexes (9,14,19). Probably, the low molecular weight polymers have reduced DNA condensation abilities, due to their reduced number of electrostatic interactions, leading to intensified aggregation at high ionic strength. At a relatively high DNA concentra- tion and a low ionic strength (150 mg/ml DNA in HBG), again small particles were formed, though the complexes were somewhat bigger than formed at lower DNA concentration (Table II). The DNA binding ability of the different p(DMAEA)- ppz_s was determined by measuring the fluorescence remain- ing upon addition of the different polymers to EtBr labeled