Structure and Dynamics of Lipoplex Formation Examined Using Two-Photon Fluorescence Cross-Correlation Spectroscopy

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Abstract
The conditions required to form transfectable lipoplexes have been extensively studied [Zuhorn, I. S., and Hoekstra, D. (2002) J. Membr. Biol.189, 167−179]. However, to date, experiments have not addressed either the order of events of lipoplex formation in solution or the maximum number of DNA molecules per vesicle in stable single-vesicle lipoplexes. In this study, we have employed two-photon excitation fluorescence correlation spectroscopy (TPE-FCS) and two-photon fluorescence cross-correlation spectroscopy (TPE-XCS) to examine both fluorescence-labeled DNA and cationic vesicle structure and dynamics simultaneously. The dependence of large aggregated lipoplex formation on DNA-to-cationic lipid charge ratio was determined, as was the maximum number of 40 bp double-stranded DNA oligonucleotides able to bind to a single vesicle.