Effect of Polymer Molecular Weight to Tumor Cells 1573 Fig. 1. Structure of poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz). polyplexes, but also polyplex formation and cytotoxicity. Moreover, polymer molecular weight was found to affect the pharmacokinetics of the polyplexes (9,13,14). For a better understanding of how polymer molecular weight affects transfection activity, these studies are of limited use as the polymers were poorly characterized, had a broad molecular weight distribution, or varied in additional characteristics like branching and conjugation with shielding or targeting li- gands. Moreover, several studies included only two different molecular weights, or focused on parameters other than transfection activity. To our knowledge, only few studies have related polymer molecular weight and in vitro transfec- tion activity, using a series of polymers with narrow molecular weight distributions (18–21). None of these studies addressed the additional issue of intravenous administration and/or in vivo tumor transfection. In this study, we used size exclusion chromatography to fractionate a batch of p(DMAEA)-ppz with a broad molec- ular weight distribution. We obtained four fractions with dif- ferent molecular weights and relatively narrow molecular weight distributions. Using these fractions, we were able to assess the effect of polymer molecular weight on the trans- fection efficiency/toxicity ratio of p(DMAEA)-ppz poly- plexes for in vitro and in vivo tumor transfection. MATERIALS AND METHODS Materials The plasmid used in this study, pcDNA3Luc, consists of the coding region for firefly luciferase inserted in the plasmid pcDNA3 (Invitrogen, UK) and is under the transcriptional con- trol of the cytomegalovirus immediate promoter. Endotoxin- free pcDNA3Luc was produced by Plasmid Factory (Bielefeld, Germany). An ethidium bromide (EtBr) stock solution was obtained from Sigma (Zwijndrecht, The Netherlands). Lucifer- ase assay reagent, reporter gene lysis buffer, and Quantilum recombinant luciferase were obtained from Promega (Leiden, The Netherlands). Polymer Synthesis Pol y(2-dimethyl amino eth yl ami no)phosphazene (p(DMAEA)-ppz) was synthesized and purified as previously described (22,23). In brief, poly(dichloro)phosphazene was syn- thesized from hexachlorocyclotriphosphazene at 250-C in 1,2,4, trichlorobenzene using sulfamic acid and CaSO42H2O as an initiating system (24). Next, the formed poly(dichloro- phosphazene) was substituted with 2-dimethylamino ethyl- amine. The resulting polymer was purified by extensive dialysis against water and THF, and the solvent was removed under reduced pressure. Finally, the polymer was dissolved in water and collected after freeze-drying. Preparative SEC Fractionation A batch of the starting polymer p(DMAEA)-ppz with a broad molar mass distribution was fractionated using a size exclusion chromatography (SEC) system. The SEC system consisted of a Waters 600 EF pump, connected to a Waters 2700 sample manager. A semi-preparative column set (Shodex OHpak SB-LG, OHpak SB-2006 M) was used. The eluent was 0.3 M NaAc (pH 5.0) with a flow rate of 1.5 ml/min. This eluent suppresses ionic interactions between the polymer and the stationary phase (25). For each fractionation run, 500 ml of solution of p(DMAEA)-ppz (10 mg/ml in eluent) was injected onto the column. Ten fractions were collected at 2 min intervals. The corresponding fractions from 400 repeated runs were pooled, extensively dialyzed against water and collected after freeze- drying. From the resulting ten fractions, two high molecular weight fractions were selected (ppz950 and ppz570) and used for experimental evaluations. Two fractions with intermedi- ate molecular weight were dissolved, pooled and subjected to a second fractionation, using the same SEC settings as de- scribed above. To this end, five fractions were collected at 3 min intervals, dialyzed and freeze-dried. From the resulting fractions, two low molecular weight fractions (ppz290 and ppz130) were selected for further evaluations. Characterization of the Polymer Fractions The average molecular weights and the molecular weight distribution of the fractionated polymers were determined by SEC, using a Viscotek VE 2001 system (25). Two Shodex SB- 804 M columns with a pre-column (Shodex OH-pak SB-G) were connected to a Triple Detector Array 302, equipped with a low and a right angle light scattering detector, a viscometer detector and a refractive index detector. The eluent was 0.3 M NaAc (pH 4.4) with a flow-rate of 1.0 ml/min. The refractive index increment (dn/dc) of p(DMAEA)-ppz was determined by injecting p(DMAEA)-ppz solutions with different concen-