Journal of Controlled Release 60 (1999) 343–353 Effect of DNA topology on the transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)–plasmid complexes J.-Y. Cherng, N.M.E. Schuurmans-Nieuwenbroek,W. Jiskoot, H. Talsma, * N.J. Zuidam, W.E. Hennink, D.J.A. Crommelin Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences ( UIPS), Utrecht University, P .O. Box 80.082, 3508 TB Utrecht, The Netherlands Received 26 December 1998; accepted 30 March 1999 Abstract In this paper the effect of the topology of plasmid DNA (supercoiled, open-circular and linear) on its binding characteristics with the polymeric transfectant poly((2-dimethylamino)ethyl methacrylate) was studied. The formed polyplexes were also evaluated for their transfection properties in vitro in two different cell lines. Anion-exchange chromatography was used for the separation of supercoiled and open-circular plasmid from a plasmid stock solution. Linear plasmids were prepared by endonucleases that cleaved the plasmid either in the promoter region or in a region not specific for expression (ampicillin resistance region). Plasmid DNA was also heat-denatured for 6 h at 708C, resulting in DNA mainly in the open-circular and oligomeric forms. The transfection of two different cell lines was dependent on the topology of the DNA in the order supercoiled.open-circular.heat-denatured.linear DNA prepared by cleaving in the nonspecific region.linear DNA prepared by cleaving in the promoter region. No differences in the size of the complexes or in the quenching of the DNA-intercalating fluorophore acridine orange were found as function of the topology. However, circular dichroism spectroscopy revealed differences between the topological plasmid species, both in the free form and in the presence of excess of cationic polymer.  1999 Elsevier Science B.V. All rights reserved. Keywords: Cationic polymer; Gene therapy; Circular dichroism spectroscopy; Fluorescence spectroscopy; Agarose gel electrophoresis; Physico–chemical characterization 1. Introduction mainly focused on structures composed of cationic lipids or polymers [2–5]. When these systems are Viral and nonviral gene delivery systems are being complexed with plasmid DNA, they are called widely investigated for the potential treatment or lipoplexes or polyplexes, respectively. The positive prevention of genetic and acquired diseases [1]. The net charge of such complexes facilitates the entry investigation of nonviral gene delivery systems is into (negatively charged) host cells, which most likely takes place via endocytosis. Finally, the DNA must end up in the nucleus for transfection to take *Corresponding author. Tel.: 131-30-253-6973; fax: 131-30- place. Besides masking of the unfavorable negative 251-7839. charge of plasmid DNA [6], binding of polycationic E-mail address: D.J.A.Crommelin@pharm.uu.nl (D.J.A. Crom- melin) systems to plasmids also leads to condensation of the 0168-3659 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0168-3659(99)00089-9