Multicomponent Cationic Lipid-DNA Complex Formation: Role of Lipid Mixing Giulio Caracciolo,* ,† Daniela Pozzi, † Heinz Amenitsch, ‡ and Ruggero Caminiti † Dipartimento di Chimica, Universita ` degli Studi di Roma “La Sapienza”, Rome, Italy 00185, and Institute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Graz, Austria Received July 29, 2005. In Final Form: October 12, 2005 Multicomponent cationic lipid-DNA complexes (lipoplexes) were prepared by adding linear DNA to mixed lipid dispersions containing two populations of binary cationic liposomes and characterized by means of small angle X-ray scattering (SAXS). Four kinds of cationic liposomes were used. The first binary lipid mixture was made of the cationic lipid (3′[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) and the neutral helper lipid dioleoylphosphocholine (DOPC) (DC-Chol/DOPC liposomes), the second one of the cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the neutral dio- leoylphosphatidylethanolamine (DOPE) (DOTAP/DOPE liposomes), the third one of DC-Chol and DOPE (DC-Chol/DOPE liposomes), and the fourth one of DOTAP and DOPC (DOTAP/DOPC liposomes). Upon DNA-induced fusion of liposomes, large lipid mixing at the molecular level occurs. As a result, highly organized mixed lipoplexes spontaneously form with membrane properties intermediate between those of starting liposomes. By varying the composition of lipid dispersions, different DNA packing density regimes can also be achieved. Furthermore, occurring lipid mixing was found to induce hexagonal to lamellar phase transition in DOTAP/DOPE membranes. Molecular mechanisms underlying experimental findings are discussed. 1. Introduction Cationic lipid-DNA complexes, named lipoplexes in the scientific community, are extensively used for cell trans- fection in vitro and are also promising candidates for in vivo gene therapy. 1-3 Lipoplexes mimic natural viruses in their ability to act as carriers of DNA, the main advantages being ease of production and potential for transfecting large pieces of DNA into cells. 1-3 Lipoplexes form spontaneously when adding DNA to dispersions of binary cationic liposomes (CLs, closed lipid bilayer shells of cationic and neutral “helper” lipids). 4 By using a plethora of experimental techniques, a variety of structures were observed with topology con- trolled by the choice of the helper lipid. 5 In the multila- mellar L R C phase, DNA chains are condensed between opposing cationic lipid membranes in the liquid-crystalline L R phase, whereas the inverted hexagonal H II C phase is comprised of lipid-coated DNA strands arranged on a hexagonal lattice. 5-7 Despite the relevant contribution clarifying the struc- ture and morphology of lipoplexes, 4-7 little is known about the mechanisms of formation. This lack of knowledge is essentially due to the high complexity of the self-assembly process. 8-10 Nevertheless, it is well accepted that decoding the underlying molecular mechanisms may yield new therapeutic means for a large quantity of disorder. 11 In this paper, we looked at the simultaneous interaction of two populations of CLs with linear DNA. CL-DNA complexes were characterized by means of high-resolution synchrotron small angle scattering (SAXS). Here we show that, upon lipoplex formation, a large lipid mixing occurs. Lipid mixing occurring during DNA-induced fusion of single species dipalmitoylphosphatidylcholine (DPPC) vesicles has been recently reported. 12 Nevertheless, as far as we know, no evidence of lipid mixing upon DNA- induced fusion of very different liposomic formulations has been provided so far. Here we also identified lipid mixing as the driving force for phase transitions in nonlamellar lipid systems. Upon complexation, multicomponent lipoplexes emerge with highly specific physical properties. It was an at- tractive result. Indeed, the engineering of multicom- ponent lipoplexes, incorporating the specific properties of very different lipid species, may represent the start- ing point to rationally design highly specific gene vectors. 13-15 To this end, we chose cationic liposomes exhibiting different lipid headgroups and a number of systematic * To whom correspondence should be addressed. E-mail: g.caracciolo@caspur.it. † Universita ` degli Studi di Roma “La Sapienza”. ‡ Austrian Academy of Sciences. (1) Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413. (2) Felgner, P. L. Sci. Am. 1997, 276, 102. (3) Lasic, D. D Liposomes in Gene Delivery; CRC Press: Boca Raton, FL, 1997. (4) Salditt, T.; Koltover, I.; Ra ¨ dler, J. O.; Safinya, C. R. Phys. Rev. Lett. 1997, 79, 2582. (5) Koltover, I.; Salditt, T.; Ra ¨ dler, J. O.; Safinya, C. R. Science 1998, 281, 78. (6) Koltover, I.; Salditt, T.; Safinya, C. R. Biophys. J. 1999, 77, 915. (7) Artzner, F.; Zantl, R.; Rapp, G.; Ra ¨dler, J. O. Phys. Rev. Lett. 1998, 81, 5015. (8) Huebner, S.; Battersby, B. J.; Grimm, R.; Cevc, G. Biophys. J. 1999, 76, 3158. (9) Barreleiro, P. C. A.; May, R. P.; Lindman, B. Faraday Discuss. 2002, 122, 191. (10) Barreleiro, P. C. A.; Lindman, B. J. Phys. Chem. B 2003, 107, 6208. (11) Kinnunen, P. K. J.; Holopainen, J. M. Bioscience Rep. 2000, 6, 465. (12) Hayes, M. E.; Gorelov, A. V.; Dawson, K. A. Prog. Colloid Polym. Sci. 2001, 118, 243. (13) Rubanyi, G. M. Mol. Asp. Med. 2001, 22, 113. (14) Prata, C. A. H.; Zhau, Y.; Barthelemy, P.; Li, Y.; Luo, D.; McIntosh, T. J.; Lee, S. J.; Grinstaff, M. W. J. Am. Chem. Soc. 2004, 1126, 12196. (15) McManus, J.; Ra ¨dler, J. O.; Dawson, K. A. J. Am. Chem. Soc. 2004, 126, 15966. 11582 Langmuir 2005, 21, 11582-11587 10.1021/la052077c CCC: $30.25 © 2005 American Chemical Society Published on Web 11/08/2005