A Stopped-flow Kinetic Study of the Assembly of Nonviral Gene Delivery Complexes
Chad S Braun 1, Mark T Fisher 2, Donald A Tomalia 3, Gary S Koe 4, Janet G Koe 4 and C. Russell Middaugh 1*
1 University of Kansas
2 University of Kansas Medical Center
3 Dendritic Nanotechnologies
4 Valentis, Inc.
* To whom correspondence should be addressed. E-mail: middaugh{at}ku.edu.
Submitted on October 28, 2004
Revised on December 21, 2004
Accepted on 25 March 2005
 |
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 CD measurements suggests complexation occurs within 50 msec. 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 which are assigned to binding/dehydration and condensation events. The rates of each process were determined over the temperature range of 10 to 50°C and activation energies were determined from the slope of Arrhenius plots. The behavior of PAMAM 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 which 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.
Key Words:
Cationic lipids, DNA, Dendrimers, Kinetics, Stopped-flow CD, Stopped-flow fluorescence
