Structure and Dynamics of Lipoplex Formation Examined Using Two-Photon Fluorescence Cross-Correlation Spectroscopy † Dennis Merkle, ‡,§ Susan P. Lees-Miller, § and David T. Cramb* ,‡ Department of Chemistry, UniVersity of Calgary, 2500 UniVersity DriVe, NW, Calgary, Alberta T2N 1N4, Canada, and Department of Biochemistry and Molecular Biology, UniVersity of Calgary, 3300 Hospital DriVe, Calgary, Alberta T2N 4N1, Canada ReceiVed NoVember 26, 2003; ReVised Manuscript ReceiVed April 7, 2004 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. While viral gene therapy possesses promising clinical application, with high transfection efficiencies, the possibility of unwanted immunological responses and mutagenesis has stimulated investigations of nonviral gene delivery methods. The dominant nonviral delivery vehicles for gene therapy are polymers and cationic lipids. Favorable electrostatic interactions drive complex formation between the DNA and lipids or polymers to form lipoplexes and polyplexes, respectively. Using appropriate lipoplex assemblies, the charged surface of the cationic lipid vesicles also ensures interaction with the negatively charged target cell surface. This step is critical for successful cellular entry of the lipoplex, which is the initial step in transfection. Lipoplexes form when counterions from the lipid surface are displaced by DNA and either the lipid vesicle collapses onto the DNA or vice versa. Various studies examining the mechanisms of association between cationic lipids and DNA have revealed a variety of factors which can be manipulated to give rise to a variety of supramolecular structures (1). Such factors include the fluidity of the cationic lipids, molar charge ratios between DNA (negative) and lipids (positive), the order of component addition (i.e., DNA to lipids or lipids to DNA), and the presence of helper lipids, salts, and other cellular components. Conditions that promote the formation of large lipoplex aggregates have been shown to possess higher transfection activity (2). Ensuring the proper mixing of the components in the presence of a positive molar charge ratio, of cationic lipid to DNA, promotes the formation of such transfectionally active lipoplex aggregates (3). There has been a wide range of studies examining the structure and formation of transfectable lipoplexes, including calorimetric analysis (4), cryoelectron imaging (5, 6), and dynamic light scattering (4). Unfortunately, such studies may be limited due to the fact that they often require conditions that are not physiologically relevant. These previous studies have also failed to elucidate exactly how much DNA is capable of lipid interaction during lipoplex formation. Fluorescence techniques provide an excellent alternative due to the fact that they can be used under physiological conditions, while the individual labeling of DNA and lipid allows for dynamic and time-resolved examination of their interaction in solution. Recently, studies using fluorescence resonance energy transfer (FRET) 1 (7) and fluorescence correlation spectros- copy (FCS) (8, 9) have examined DNA interactions with both polymers (7-9) and lipids (LipofectAMINE) (7). These studies were limited by the fact that they monitored a single species at a time. As an alternative, two-photon excitation † This work was funded through operating grants from the Natural Sciences and Engineering Research Council and the Canadian Institute for Photonics Innovation (to D.T.C.) and the National Cancer Institute of Canada and the Alberta Cancer Board (to S.P.L.-M.). S.P.L.-M. is a Heritage Medical Scientist of the Alberta Heritage Foundation for Medical Research and an Investigator of the Canadian Institutes for Health Research and holds the Alberta Cancer Foundation/Engineered Air Chair in Cancer Research. D.M. was supported by a studentship through the Alberta Cancer Board. * To whom correspondence should be addressed: Department of Chemistry, University of Calgary, 2500 University Dr., NW, Calgary, AB T2N 1N4, Canada. Phone: (403) 220-8138. Fax: (403) 289-9488. E-mail: dcramb@ucalgary.ca. ‡ Department of Chemistry. § Department of Biochemistry and Molecular Biology. 1 Abbreviations: CR, charge ratio; DLS, dynamic light scattering; DOTAP, 1,2-dioleoyl-3-trimethylammonium propane; DOTAPf, DOTAP labeled with fluorescein-DHPE; DOTAPl, DOTAP labeled with lissamine-DOPE; FCS, fluorescence correlation spectroscopy; fluorescein-DHPE, N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl- sn-glycero-3-phosphoethanolamine; FRET, fluorescence resonance energy transfer; ITC, isothermal calorimetry; lissamine-DOPE, lissamine dioleoylphosphatidylethanolamine; SUV, single unilamellar vesicle; TPE, two-photon excitation; XCS, fluorescence cross-correlation spectroscopy. 7263 Biochemistry 2004, 43, 7263-7272 10.1021/bi036133p CCC: $27.50 © 2004 American Chemical Society Published on Web 05/18/2004