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A Stopped-Flow Kinetic Study of the Assembly of Nonviral Gene Delivery Complexes
Braun, Chad S. Fisher, Mark T. Tomalia, Donald A. Koe, Gary S. Koe, Janet G. Middaugh, C. Russell
Braun, Chad S.
Fisher, Mark T.
Tomalia, Donald A.
Koe, Gary S.
Koe, Janet G.
Middaugh, C. Russell
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Abstract
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.
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2005-06
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Biophysical Society
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Braun et. al. "A Stopped-Flow Kinetic Study of the Assembly of Nonviral Gene Delivery Complexes." Volume 88, Issue 6, June 2005, Pages 4146–4158.
http://dx.doi.org/10.1529/biophysj.104.055202