Toward the Rational Design of Lipid Gene Vectors: Shape Coupling between Lipoplex and Anionic Cellular Lipids Controls the Phase Evolution of Lipoplexes and the Efficiency of DNA Release Daniela Pozzi,* ,† Giulio Caracciolo,* ,† Ruggero Caminiti, † Sofia Candeloro De Sanctis, † Heinz Amenitsch, ‡ Cristina Marchini,* ,§ Maura Montani, § and Augusto Amici § Department of Chemistry, University of Rome “La Sapienza”, Piazzale A. Moro 5, 00185 Rome, Italy, Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, Schmiedelstrasse 6, A-8042 Graz, Austria, and Genetic Immunization Laboratory, Department of Molecular Cellular and Animal Biology, University of Camerino, Via Gentile III da Varano, 62032 Camerino (MC), Italy ABSTRACT A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interaction with anionic cellular lipids, resulting in DNA release. At the early stages of interaction, we found a universal behavior of lipoplex/anionic lipid (AL) mixtures: the lipoplex structure is slightly perturbed, while the one-dimensional DNA lattice between cationic membranes is largely diluted by ALs. This finding is in excellent agreement with previous suggestions on the mechanism of DNA unbinding from lipoplexes by ALs. Upon further interaction, the propensity of a given lipoplex structure to be solubilized by anionic cellular lipids strongly depends on the shape coupling between lipoplex and ALs. Furthermore, we investigated the effect of the membrane charge density and a general correlation resulted: the higher the membrane charge density of anionic membranes, the higher their ability to solubilize the structure of lipoplexes and to promote DNA release. Lastly, the formation of nonlamellar phases in lipoplex/AL mixtures is regulated by the propensity of anionic cellular lipids to adopt nonlamellar phases. Remarkably, also phase transition rates and DNA release were found to be strongly affected by the shape coupling between lipoplex and ALs. It thus seems likely that the structural and phase evolution of lipoplexes may only be meaningful in the context of specific anionic cellular membranes. These results highlight the phase properties of the carrier lipid/cellular lipid mixtures as a decisive factor for optimal DNA release and suggest a potential strategy for the rational design of efficient cationic lipid carriers. KEYWORDS: cationic liposomes • DNA • lipoplexes • cellular lipids • cell transfection • small-angle X-ray scattering • gel electrophoresis 1. INTRODUCTION N owadays, synthetic cationic liposomes (CLs) are the most promising nonviral gene vectors (1). When mixed with negatively charged DNA, CLs form stable complexes (lipoplexes) with a well-ordered structure at the nanoscale (Figure 1). During the past decade, the ease of cationic lipid synthesis and the availability of lipid tech- nologies resulted in a huge number of lipid transfection reagents. Most reports have presented data on various cationic lipid chemical structures and also on formulation procedures (2). Physical-chemical properties of lipoplexes have been deeply investigated both theoretically (3-10) and experi- mentally (11-25). Unfortunately, the absence of supporting biology resulted in the “plateauing” of transfection efficien- * To whom correspondence should be addressed. E-mail: d.pozzi@caspur.it (D.P.), g.caracciolo@caspur.it (G.C.), cristina.marchini@unicam.it (C.M.). Tel.: (+39) 06 49913076, (+39) 0737 403276. Fax: (+39) 06 490631, 0737403290. Received for review June 11, 2009 and accepted September 8, 2009 † University of Rome “La Sapienza”. ‡ Austrian Academy of Sciences. § University of Camerino. DOI: 10.1021/am900406b © 2009 American Chemical Society FIGURE 1. Schematic picture of the equilibrium phases of lipoplexes, showing the local structure of their interior on the nanometer scale. Neutral and cationic lipids are depicted as having white and black headgroups, respectively. The lamellar phase, composed of alterna- tive lipid bilayers and DNA monolayers, with a repeat spacing given by d ) d B + d W is depicted on the left. The hexagonal phase, which consists of DNA coated with a lipid monolayer arranged on a hexagonal lattice, is shown on the right. ARTICLE www.acsami.org VOL. 1 • NO. 10 • 2237–2249 • 2009 2237 Published on Web 09/24/2009 Downloaded by UNIVERSITA DI CAMERINO on October 29, 2009 | http://pubs.acs.org Publication Date (Web): September 24, 2009 | doi: 10.1021/am900406b