A Columnar Phase of Dendritic Lipid-Based Cationic Liposome-DNA Complexes for Gene Delivery: Hexagonally Ordered Cylindrical Micelles Embedded in a DNA Honeycomb Lattice Kai K. Ewert, Heather M. Evans, ² Alexandra Zidovska, Nathan F. Bouxsein, Ayesha Ahmad, § and Cyrus R. Safinya* Contribution from the Department of Materials, Department of Physics, and Molecular, Cellular and DeVelopmental Biology Department, UniVersity of California, Santa Barbara, California 93106 Received August 26, 2005; E-mail: safinya@mrl.ucsb.edu Abstract: Gene therapy holds great promise as a future approach to fighting disease and is explored in worldwide clinical trials. Cationic liposome (CL)-DNA complexes are a prevalent nonviral delivery vector, but their efficiency requires improvement and the understanding of their mechanism of action is incomplete. As part of our effort to investigate the structure-transfection efficiency relationships of self-assembled CL- DNA vectors, we have synthesized a new, highly charged (16+) multivalent cationic lipid, MVLBG2, with a dendritic headgroup. Our synthetic scheme allows facile variation of the headgroup charge and the spacer connecting hydrophobic and headgroup moieties as well as gram-scale synthesis. Complexes of DNA with mixtures of MVLBG2 and neutral 1,2-dioleoyl-sn-glycerophosphatidylcholine (DOPC) exhibit the well- known lamellar phase at 90 mol % DOPC. Starting at 20 mol % dendritic lipid, however, two novel nonlamellar phases are observed by synchrotron X-ray diffraction. The structure of one of these phases, present in a narrow range of composition around 25 mol % MVLBG2, has been solved. In this novel dual lattice structure, termed H I C , hexagonally arranged tubular lipid micelles are surrounded by DNA rods forming a three-dimensionally continuous substructure with honeycomb symmetry. Complexes in the HI C phase efficiently transfect mouse and human cells in culture. Their transfection efficiency, as well as that of the lamellar complexes containing only 10 mol % dendritic lipid, reaches and surpasses that of commercially available, optimized DOTAP-based complexes. In particular, complexes containing MVLBG2 are significantly more transfectant over the entire composition range in mouse embryonic fibroblasts, a cell line empirically known to be hard to transfect. Introduction Somatic gene therapy, i.e., the use of DNA as a therapeutic agent, holds great promise for future medical applications. Indeed, numerous clinical trials in this field are currently ongoing. 1,2 Cancers, inherited diseases, cardiovascular diseases, and many others are targets for this novel medical approach. 3,4 In fact, the therapeutic prospects of nucleic acid delivery are expanding constantly 5 due to recent discoveries such as that of RNA interference (RNAi), which enables, in principle, selective gene silencing. 6-9 Thus, substantial research efforts are directed toward developing and fundamentally understanding DNA carriers (vectors). Engineered viruses are very efficient vec- tors, 10,11 but general concerns about their safety have recently been intensified by a few severe setbacks. 12 This has further increased the interest in nonviral or synthetic vectors, 13-15 in which the negatively charged DNA is complexed with cationic ² Current address: Max Planck Institute for Dynamics and Self- Organization, Bunsenstrasse 10, D-37073 Go ¨ttingen, Germany. § Current address: Imperial College Genetic Therapies Centre, Depart- ment of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London, SW7 2AZ, U.K. (1) Extensive and current information on clinical trials in the field of gene therapy can be found on the Internet at http://www.wiley.co.uk/genetherapy/ clinical/. See Ewert, K.; Ahmad, A.; Evans, H. M.; Safinya, C. R. Expert Opin. Biol. Ther. 2005, 5, 33-53 for a compilation of open trials in nonviral gene therapy as of July 2004. (2) Griesenbach, U.; Geddes, D. M.; Alton, E. W. F. W. In NonViral Vectors for Gene Therapy; Huang, L., Hung, M.-C., Wagner, E., Eds.; Academic Press: San Diego, 1999; pp 337-356. (3) Felgner, P. L.; Rhodes, G. Nature 1991, 349, 351-352. (4) Smyth-Templeton, N., Lasic, D. D., Eds. Gene Therapy. Therapeutic Mechanisms and Strategies; Marcel Dekker Inc.: New York, 2000. (5) Mahato, R. I., Kim, S. W., Eds. Pharmaceutical PerspectiVes of Nucleic Acid-Based Therapeutics; Taylor and Francis: London and New York, 2002. (6) McManus, M. T.; Sharp, P. A. Nat. ReV. Genet. 2002, 3, 737-747. (7) Caplen, N. J. Gene Ther. 2004, 11, 1241-1248. (8) Milner, J. Expert Opin. Biol. Ther. 2003, 3, 459-467. (9) Caplen, N. J. Expert Opin. Biol. Ther. 2003, 3, 575-586. (10) Smith, A. E. Annu. ReV. Microbiol. 1995, 49, 807-838. (11) Kay, M. A.; Glorioso, J. C.; Naldini, L. Nature Med. 2001, 7, 33-40. (12) (a) Marshall, E. Science 2000, 288, 951-957. (b) Marshall, E. Science 2002, 298, 510-511. (13) Huang, L., Hung, M.-C., Wagner, E., Eds. Non-Viral Vectors for Gene Therapy, 2nd ed., Part I; Advances in Genetics, Vol. 53; Elsevier: San Diego, 2005. (14) Huang, L., Hung, M.-C., Wagner, E., Eds.; NonViral Vectors for Gene Therapy; Academic Press: San Diego, 1999. (15) Felgner, P. L., Heller, M. J., Lehn, P., Behr, J.-P., Szoka, F. C., Eds.; Artificial Self-Assembling Systems for Gene DeliVery; American Chemical Society: Washington, DC, 1996. Published on Web 03/08/2006 3998 9 J. AM. CHEM. SOC. 2006, 128, 3998-4006 10.1021/ja055907h CCC: $33.50 © 2006 American Chemical Society