Pharmaceutical Research, Vol. 24, No. 8, August 2007 ( # 2007) DOI: 10.1007/s11095-007-9299-z Research Paper Biodegradable Poly(2-Dimethylamino Ethylamino)Phosphazene for In Vivo Gene Delivery to Tumor Cells. Effect of Polymer Molecular Weight Holger K. de Wolf,1 Markus de Raad,1 Cor Snel,1 Mies J. van Steenbergen,1 Marcel H. A. M. Fens,1 Gert Storm,1 and Wim E. Hennink1,2 Received January 5, 2007; accepted March 16, 2007; published online April 11, 2007 Purpose. Previously, we have shown that complexes of plasmid DNA with the biodegradable polymer poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) mediated tumor selective gene expression after intravenous administration in mice. In this study, we investigated the effect of p(DMAEA)-ppz molecular weight on both in vitro and in vivo tumor transfection, as well as on complex induced toxicity. Materials and Methods. p(DMAEA)-ppz with a broad molar mass distribution was fractionated by preparative size exclusion chromatography. Polyplexes consisting of plasmid DNA and the collected polymer fractions were tested for biophysical properties, (cyto)toxicity and transfection activity. Results. Four p(DMAEA)-ppz fractions were collected with weight average molecular weights ranging from 130 to 950 kDa, and with narrow molecular mass distributions (Mw/Mn from 1.1 to 1.3). At polymer-to-DNA (N/P) ratios above 6, polyplexes based on these polymers were all positively charged (zeta potential 25–29 mV), and had a size of 80–90 nm. The in vitro cytotoxicity of the polyplexes positively correlated with polymer molecular weight. The in vitro transfection activity of the different polyplexes depended on their N/P ratio, and was affected by the degree of cytotoxicity, as well as the colloidal stability of the different polyplexes. Intravenous administration of polyplexes based on the high molecular weight polymers led to apparent toxicity, as a result of polyplex-induced erythrocyte aggregation. On the other hand, administration of polyplexes based on low molecular weight p(DMAEA)-ppz_s (Mw 130 kDa) did not show signs of toxicity and resulted in tumor selective gene expression. Conclusion. Polymer molecular weight fractionation enabled us to optimize the transfection efficiency/ toxicity ratio of p(DMAEA)-ppz polyplexes for in vitro and in vivo tumor transfection. KEY WORDS: biodegradable; cationic polymer; DNA; molecular weight; tumor gene delivery. INTRODUCTION Non-viral gene delivery holds promise as a therapy against cancer. The genes used for application in anticancer gene med- icine encode for antigens, cytokines and suicide proteins (1). In order to efficiently reach metastatic tumors, intravenous admin- istration is the preferred route of administration. In this light, cationic polymers can be useful. The condensation of plasmid DNA using cationic polymers results in positively charged, nanosized polyplexes with improved resistance against nucleases present in blood and other extracellular fluids. In addition, positively charged polyplexes are readily taken up by the target cells. Moreover, surface modification of polyplexes enables improved circulation kinetics and tumor targeting. Indeed, the application of polymers for cancer gene therapy so far has resulted in tumor regression in several animal models (1,2). 1 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 80082, 3508 TB, Utrecht, The Netherlands. 2 To whom correspondence should be addressed. (e-mail: W.E.Hennink@ pharm.uu.nl) Cationic polymers currently used for gene delivery are mostly non-degradable and cytotoxic, limiting their wide- spread use. The application of biodegradable polymers in polyplexes is a means to prevent long-term polymer tissue ac- cumulation and therefore limit toxicity (3). Ideally, the deg- radation of the polymer will result in low molecular weight fragments which will be subjected to glomerular kidney filtration or will be metabolized (4). Recently, we reported on the application of the biodegradable poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) for tumor gene delivery (Fig. 1). It was shown that polyplexes based on p(DMAEA)-ppz mediated gene expression to a similar extent as linear polyethylenimine (PEI). Gene expression was con- fined to the tumor tissue, whereas the use of PEI polyplexes also resulted in substantial expression in non-target organs, particularly in lungs, spleen and liver (5). Biodegradable as well as non-biodegradable gene deliv- ery polymers generally have a broad molecular weight dis- tribution. Several studies have reported on the importance of polymer molecular weight in the outcome of transfection (6– 17). From these studies, it appears that polymer molecular weight not only affects the transfection efficiency of the 0724-8741/07/0800-1572/0 # 2007 Springer Science + Business Media, LLC 1572