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A Stopped-Flow Kinetic Study of the Assembly of Nonviral Gene Delivery Complexes

Chad S. Braun ^*, Mark T. Fisher , Donald A. Tomalia , Gary S. Koe , Janet G. Koe  and C. Russell Middaugh ^*

^* Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047; Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160; Dendritic Nanotechnologies, Mt. Pleasant, Michigan 48858; and Valentis Inc., Burlingame, California 94010

Correspondence: Address reprint requests to C. Russell Middaugh, Dept. of Pharmaceutical Chemistry, 2095 Constant Ave., Lawrence, KS 66047. Tel.: 785-864-5813; Fax: 785-864-5814; Email: middaugh{at}ku.edu.

Stopped-flow circular dichroism and fluorescence spectroscopy are used to characterize the assembly of complexes consisting of plasmid DNA bound to the cationic lipids dimethyldioctadecylammonium bromide and 1, 2-dioleoyl- 3-trimethylammonium-propane and a series of polyamidoamine dendrimers. The kinetics of complexation determined from the stopped-flow circular dichroism measurements suggests complexation occurs within 50 ms. Further analysis, however, was precluded by the presence of mixing (shear) artifacts. Stopped-flow fluorescence employing the high-affinity DNA dyes Hoechst 33258 and YOYO-1 was able to resolve two sequential steps in the assembly of complexes that are assigned to binding/dehydration and condensation events. The rates of each process were determined over the temperature range of 10–50°C and activation energies were determined from the slope of Arrhenius plots. The behavior of polyamidoamine dendrimers can be separated into two classes based on their differing binding modes: generation 2 and the larger generations (G4, G7, and G9). The larger generations have activation energies for binding that follow the trend G4 > G7 > G9. The activation energies for condensation (compaction) of complexes composed of these same dendrimers have the opposite trend G9 > G7 > G4. It is postulated that a balance between a more energetically favorable condensation and less favorable binding may prove beneficial in enhancing gene delivery.