Abstract Cationic liposomes/DNA complexes are wide- ly used vectors for delivering genes in clinical and ex- perimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposo- mal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vit- ro were verified in vivo using a porcine restenosis mod- el. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maxi- mum transfer efficiency were firstly the appropriate se- lection of cationic lipids for the cell type to be transfec- ted, secondly the DNA/liposome ratio chosen, which de- pended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold high- er transfer efficiencies when transfer conditions were ex- trapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ß-galacto- sidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer effi- ciencies, gene expression, and biological effect. Station- ary cell cultures simulate more realistically the in vivo J. Pelisek · S. Nikol ( ✉ ) Department of Cardiology and Angiology, University Hospital, Westfälische Wilhelm University, 48129 Münster, Germany e-mail: nikol@uni-muenster.de Tel.: +49-251-8348501, Fax: +49-251-8345101 J. Pelisek · M.G. Engelmann · A. Golda · S. Armeanu M. Shimizu · S. Nikol Medical Department I, Klinikum Großhadern, Ludwig Maximilian University, Munich, Germany J. Pelisek Department of Pharmacy, Center of Drug Research, Pharmaceutical Biology-Biotechnology, Ludwig Maximilian University, Munich, Germany A. Fuchs Pediatric Clinic, Klinikum Grosshadern, Ludwig Maximilian University, Munich, Germany C. Mekkaoui · P.H. Rolland Laboratoire Cardiovasculaire, Faculté de Médécine, Université de Marseille, France J Mol Med (2002) 80:724–736 DOI 10.1007/s00109-002-0368-9 ORIGINAL ARTICLE Jaroslav Pelisek · Markus G. Engelmann Adam Golda · Alexandra Fuchs · Sorin Armeanu Masumi Shimizu · Choukri Mekkaoui Pierre H. Rolland · Sigrid Nikol Optimization of nonviral transfection: variables influencing liposome-mediated gene transfer in proliferating vs. quiescent cells in culture and in vivo using a porcine restenosis model Received: 12 February 2002 / Accepted: 1 July 2002 / Published online: 24 October 2002 © Springer-Verlag 2002 SIGRID NIKOL received her training at the University of Aachen, Tufts University, Boston (J.M. Isner), and Ludwig Maximilan University, Munich. She was appointed Associate Professor for Molecular Cardiology at the University of Münster in 2001. Her major research in- terests include gene therapy and local drug delivery. JAROSLAV PELISEK received his Ph.D. in biologi- cal sciences from Ludwig Maximilian University, Mu- nich. His research interests in- clude the optimization of non- viral vector systems in vitro and in a porcine restenosis model. His current research field is the active transport of transfected DNA into the nu- cleus of nondividing cells.