A Theoretical and Experimental Approach to the Compaction Process of DNA by Dioctadecyldimethylammonium Bromide/Zwitterionic Mixed Liposomes Alberto Rodrı ´guez-Pulido, † Alberto Martı ´n-Molina, ‡ Ce ´sar Rodrı ´guez-Beas, ‡ Oscar Llorca, § Emilio Aicart, † and Elena Junquera* ,† Grupo de Quı ´mica Coloidal y Supramolecular, Departamento de Quı ´mica Fı ´sica I, Facultad de Ciencias Quı ´micas, UniVersidad Complutense de Madrid, 28040-Madrid, Spain, Grupo de Fı ´sica de Fluidos y Biocoloides, Departamento de Fı ´sica Aplicada, Facultad de Ciencias, UniVersidad de Granada 18071- Granada, Spain, and Centro de InVestigaciones Biolo ´gicas, CSIC, Ramiro de Maeztu 9, 28040-Madrid, Spain ReceiVed: July 17, 2009; ReVised Manuscript ReceiVed: September 9, 2009 The compaction of DNA by cationic liposomes constituted by a mixture of a cationic lipid, dioctadecyldim- ethylammonium bromide (DODAB), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanol- amine (DOPE) or 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), has been evaluated by means of experimental studies (electrophoretic mobility, conductometry, cryogenic electron transmission microscopy or cryo-TEM, and fluorescence spectroscopy) as well as theoretical calculations. This information reveals that DODAB/DOPE and DODAB/DLPC liposomes are mostly spherical and unilamellar, with a mean diameter of around 70 and 61 nm, respectively, a bilayer thickness of 4.5 nm, and gel-to-fluid transition temperatures, T m , of around 19 and 28 °C, respectively. Their positively charged surfaces efficiently compact the negatively charged DNA by means of a strong entropically driven surface interaction that yields DODAB/DOPE-DNA and DODAB/DLPC-DNA lipoplexes as confirmed by zeta potential and ethidium bromide fluorescence intercalation assays. These experiments have permitted as well the evaluation of the different microenvironments of varying polarity of the DNA helix, liposomes, and/or lipoplexes. DODAB/DOPE-DNA and DODAB/ DLPC-DNA lipoplexes have been characterized by isoneutrality ratios (L/D) φ of around 4.7 and 4.8, respectively, a more fluid membrane than that of the parent liposomes, and T m around 24 and 28 °C, respectively, as revealed by fluorescence anisotropy. Cryo-TEM micrographs reveal a rich scenario of nanostructures and morphologies, from unilamellar DNA-coated liposomes to multilamellar lipoplexes passing through cluster-like structures. Phase diagrams (aggregation and re-entrant condensation phenomena), calculated by means of a phenomenological theory, have confirmed the experimental concentration domains and the isoneutrality conditions. The influence of helper lipid in the compaction process, as well as the optimum choice among those herein chosen, has been analyzed. 1. Introduction Although viral-based DNA carriers constitute nowadays the most common method for gene delivery, there has been in the past decade increasing research in developing nonviral vectors. 1-8 Particularly, cationic liposomes have shown potential application as transfer vectors, because of their facile production and low toxicity. 4,5,9-12 The transmission efficiency of DNA using cationic liposomes (CL) varies so much depending on the kind of liposome, the presence of helper lipid, the lipid/DNA ratio, or the cells targeted, 4,6,13 although a universal behavior can be obtained when liposome characteristics are compared with respect to charge density. 14,15 From those previous studies, it is clear that the interaction between cationic liposomes and DNA, when forming the complex named lipoplex, plays an important role, and for that reason a currently renovated area of research is focused on improving the understanding of this interaction, from both experimental and theoretical points of view. 6,13 In fact, it is expected that a physicochemical characterization of the process can shed light on the formation of these complexes and, accordingly, on transfection mechanisms. It is well known that the presence of both a cationic and a neutral helper lipid, forming a mixed liposome, may improve the efficiency of transfection. 4,6,13 In fact, the choice of the helper is very important because, depending on it, the transfection efficiency can be increased or unaffected. 16 We recently reported 17,18 a series of experimental studies of DNA compaction with mixed liposomes constituted by a cationic lipid and a zwitterionic helper lipid of the phosphatidylethanolamine family (DOPE). The results revealed mainly two features: (a) a strong surface electrostatic interaction drives the formation of li- poplexes; (b) lipoplexes showed, in the vicinity of isoneutrality, a clear tendency to form cluster-type structures not well understood. With the aim of shedding light on these and others points related to lipoplexes, we report in this work a deeper study involving lipoplexes formed by DNA with the cationic lipid DODAB (a dialkyldimethylammonium cationic lipid with two 18C saturated hydrocarbon tails), in the presence of two helper zwitterionic lipids, DOPE and/or DLPC. DOPE is a helper lipid with a single olefinic unsaturation in cis configu- ration at the 9 position in the two 18C hydrocarbon chains, while DLPC, of the phosphatidylcholine family, presents full saturation in the two 12C hydrocarbon chains. Both (mostly DOPE) are * To whom correspondence should be addressed. Tel: +34-91-394-4131. Fax: +34-91-394-4135. E-mail: junquera@quim.ucm.es. http://www.ucm.es/ info/coloidal/index.html. † Universidad Complutense de Madrid. ‡ Universidad de Granada. § CSIC. J. Phys. Chem. B 2009, 113, 15648–15661 15648 10.1021/jp906777g CCC: $40.75  2009 American Chemical Society Published on Web 10/30/2009