A stopped-flow kinetic study of the assembly of nonviral gene delivery complexes.
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 degrees 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.
Braun
Chad S
CS
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, 66047, USA.
Fisher
Mark T
MT
Tomalia
Donald A
DA
Koe
Gary S
GS
Koe
Janet G
JG
Middaugh
C Russell
CR
D001583:Benzoxazoles; D055592:Biophysical Phenomena; D001703:Biophysics; D006690:Bisbenzimidazole; D002412:Cations; D002942:Circular Dichroism; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D016503:Drug Delivery Systems; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D011806:Quinolinium Compounds; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001583:Benzoxazoles; D002412:Cations; D005456:Fluorescent Dyes; D008081:Liposomes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA; D006690:Bisbenzimidazole