DNA/Fusogenic Lipid Nanocarrier Assembly: Millisecond Structural Dynamics Borislav Angelov, † Angelina Angelova,* ,‡ Sergey K. Filippov, † Theyencheri Narayanan, § Markus Drechsler, ⊥ Petr S ̌ tě pa ́ nek, † Patrick Couvreur, ‡ and Sylviane Lesieur ‡ † Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, CZ-16206 Prague, Czech Republic ‡ CNRS UMR8612 Institut Galien Paris-Sud, Univ Paris Sud 11, F-92296 Châ tenay-Malabry, France § European Synchrotron Radiation Facility (ESRF), 6 rue Jules Horowitz, F-38043 Grenoble, France ⊥ Laboratory for Soft Matter Electron Microscopy, Bayreuth Institute of Macromolecular Research, University of Bayreuth, D-95440 Bayreuth, Germany * S Supporting Information ABSTRACT: Structural changes occurring on a millisecond time scale during uptake of DNA by cationic lipid nanocarriers are monitored by time-resolved small-angle X-ray scattering (SAXS) coupled to a rapid-mixing stopped-flow technique. Nanoparticles (NPs) of nanochannel organization are formed by PEGylation, hydration, and dispersion of a lipid film of the fusogenic lipid monoolein in a mixture with positively charged (DOMA) and PEGylated (DOPE-PEG 2000 ) amphiphiles and are characterized by the inner cubic structure of very large nanochannels favorable for DNA upload. Ultrafast structural dynamics of complexation and assembly of these cubosome particles with neurotrophic plasmid DNA (pDNA) is revealed thanks to the high brightness of the employed synchrotron X-ray beam. The rate constant of the pDNA/lipid NP complexation is estimated from dynamic roentgenograms recorded at 4 ms time resolution. pDNA upload into the vastly hydrated channels of the cubosome carriers leads to a fast nanoparticle-nanoparticle structural transition and lipoplex formation involving tightly packed pDNA. SECTION: Biomaterials, Surfactants, and Membranes S tudies of structure and stability of lipid/DNA complexes (lipoplexes) 1-17 are of ongoing interest for the success of prospective nanoparticle-based gene therapies. In this field, small-angle X-ray scattering (SAXS) has been recognized as a powerful structural technique for the design of DNA and drug carrier systems. 3,18-28 In a pioneering work, Lindman and colleagues 6 have stressed that the mechanism governing the lipoplex formation has not been understood yet due to the lack of information on the kinetics. Toward that aim, stopped-flow turbidity, fluorescence, and small-angle neutron scattering (SANS) have been employed to study the kinetics of salmon sperm DNA/liposome complex formation. 7 SANS measure- ments have established intermediate cylindrical structures, formed from liposomes on the time scale of seconds, and multilamellar aggregates grown on the time scale of minutes. 7 However, fluorescence and turbidity measurements have shown the necessity of elucidating the nanostructures forming on the shorter time scales. 6 Therefore, the in-depth understanding of the DNA/lipid nanoparticle (NP) assembly mechanism and kinetic pathways requires micro- and millisecond time-resolved structural investigations performed at state-of-the-art X-ray sources. 29-31 Here, we report rapid-mixing stopped-flow SAXS inves- tigation, on the millisecond time scale, of the assembly and complexation between neurotrophin-encoding plasmid DNA (pDNA) and fusogenic lipid carriers with nanochannelled organization (cubosome nanoparticles). It is considered that cubic NPs, built up by nonphospholipid molecules of a nonlamellar propensity (such as the fusogenic lipid monoolein, MO), 32-36 can mimic the structural and transport properties of certain porous icosahedral virion particles (see Figure 1S, Supporting Information). The controlled NP formulation with pDNA, using millisecond microfluidic mixing, 37 is anticipated to overcome the problems related to broad size distribution, physical instability, heterogeneous membrane structures, and rigid bulky aggregate formation observed with lipoplexes. 14,17 The kinetics of cubic membrane particle/pDNA complexation, enlightening a millisecond-range nanoparticle-nanoparticle structural transition, has not been previously investigated. The presented methodology for real time monitoring of the pDNA/lipid NP assembly offers a new approach for determination of the rate constants of the complexation from micro- and millisecond time-resolved roentgenograms. PEGylated nanoparticles with nonlamellar inner-membrane organization and open nanochannels were prepared by hydration and dispersion of a mixed lipid film containing the Received: April 23, 2013 Accepted: May 21, 2013 Published: May 28, 2013 Letter pubs.acs.org/JPCL © 2013 American Chemical Society 1959 dx.doi.org/10.1021/jz400857z | J. Phys. Chem. Lett. 2013, 4, 1959-1964