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Three-Dimensional Imaging of Lipid Gene-Carriers: Membrane Charge Density Controls Universal Transfection Behavior in Lamellar Cationic Liposome-DNA Complexes

Alison J. Lin ^*, Nelle L. Slack ^*, Ayesha Ahmad ^*, Cyril X. George , Charles E. Samuel  and Cyrus R. Safinya ^*

^* Materials Department, Physics Department, and Biomolecular Science and Engineering Program, University of California, Santa Barbara, California 93106; and Molecular, Cellular, and Developmental Biology Department, and Biomolecular Science and Engineering Program, University of California, Santa Barbara, California 93106

Correspondence: Address reprint requests to C. R. Safinya, MRL Rm. 2208, University of California, Santa Barbara, CA 93106. Tel.: 805-893-8635; Fax: 805-893-7221; E-mail: safinya{at}mrl.ucsb.edu.

Cationic liposomes (CLs) are used worldwide as gene vectors (carriers) in nonviral clinical applications of gene delivery, albeit with unacceptably low transfection efficiencies (TE). We present three-dimensional laser scanning confocal microscopy studies revealing distinct interactions between CL-DNA complexes, for both lamellar L^C and inverted hexagonal H_II^C nanostructures, and mouse fibroblast cells. Confocal images of L^C complexes in cells identified two regimes. For low membrane charge density (_M), DNA remained trapped in CL-vectors. By contrast, for high _M, released DNA was observed in the cytoplasm, indicative of escape from endosomes through fusion. Remarkably, firefly luciferase reporter gene studies in the highly complex L^C-mammalian cell system revealed an unexpected simplicity where, at a constant cationic to anionic charge ratio, TE data for univalent and multivalent cationic lipids merged into a single curve as a function of _M, identifying it as a key universal parameter. The universal curve for transfection by L^C complexes climbs exponentially over four decades with increasing _M below an optimal charge density (_M^*), and saturates for at a value rivaling the high transfection efficiency of H_II^C complexes. In contrast, the transfection efficiency of H_II^C complexes is independent of _M. The exponential dependence of TE on _M for L^C complexes, suggests the existence of a kinetic barrier against endosomal fusion, where an increase in _M lowers the barrier. In the saturated TE regime, for both L^C complexes and H_II^C, confocal microscopy reveals the dissociation of lipid and DNA. However, the lipid-released DNA is observed to be in a condensed state, most likely with oppositely charged macro-ion condensing agents from the cytoplasm, which remain to be identified. Much of the observed bulk of condensed DNA may be transcriptionally inactive and may determine the current limiting factor to transfection by cationic lipid gene vectors.

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