COMMUNICATIONS Lipid Phase Control of DNA Delivery Rumiana Koynova,* ,† Li Wang, Yury Tarahovsky, ‡ and Robert C. MacDonald Biochemistry, Molecular & Cell Biology, Northwestern University, Evanston, Illinois 60208. Received July 27, 2005; Revised Manuscript Received October 5, 2005 Cationic lipids form nanoscale complexes (lipoplexes) with polyanionic DNA and can be utilized to deliver DNA to cells for transfection. Here we report the correlation between delivery efficiency of these DNA carriers and the mesomorphic phases they form when interacting with anionic membrane lipids. Specifically, formulations that are particularly effective DNA carriers form phases of highest negative interfacial curvature when mixed with anionic lipids, whereas less effective formulations form phases of lower curvature. Structural evolution of the carrier lipid/DNA complexes upon interaction with cellular lipids is hence suggested as a controlling factor in lipid-mediated DNA delivery. A strategy for optimizing lipofection is deduced. The behavior of a highly effective lipoplex formulation, DOTAP/DOPE, is found to conform to this “efficiency formula”. Efficient delivery of genetic material into biological systems is needed for tasks of utmost importance in laboratory and clinic, such as gene transfection and gene silencing. Synthetic cationic lipids can be used as DNA carriers and are now considered the most promising nonviral gene carriers (1). They form complexes (lipo- plexes) with polyanionic DNA. Understanding the mech- anism of lipid-mediated DNA delivery (lipofection) is of paramount significance for their effective application, as well as for rational design and synthesis of novel cationic lipid compounds that are promising for superior gene delivery. Here we advance the concept that the control- ling factor in lipofection is the structural evolution of lipoplexes upon interaction with cellular (anionic) lipids. The unbinding of DNA from a cationic lipid carrier when the lipoplex gets inside the cell has been identified as one of the key steps in lipofection. According to the current understanding, the unbinding is a result of charge neutralization by cellular anionic lipids; indeed, experiments have revealed that addition of negatively charged liposomes to lipoplexes results in dissociation of DNA from the lipid (2-6). A set of significant recent findings suggest that the structure of cationic lipid carriers changes dramatically upon interaction with cellular lipids, and furthermore that such changes may critically affect the delivery efficiency. Hydrated lipids are known for their ability to form an impressive variety of polymorphic and mesomorphic phases, lamellar and nonlamellar, brought about by the optimization of the hydrophobic effect in conjunction with various intra- and intermolecular interactions and geo- metric packing constraints (7, 8). These phases are generally differentiated by the curvature of their lipid- water interface. The lipid phase state is a key physical * Corresponding author. E-mail: r-tenchova@northwestern.edu. † Associate member of the Institute of Biophysics, Bulgarian Academy of Sciences. ‡ Current address: Institute of Theoretical and Experimental Biophysics, 142290, Pushchino, Russia. NOVEMBER/DECEMBER 2005 Volume 16, Number 6 © Copyright 2005 by the American Chemical Society 10.1021/bc050226x CCC: $30.25 © 2005 American Chemical Society Published on Web 10/20/2005