To evaluate
complex stability in serum by agarose gel electrophoresis, lead carriers
(4–50, 6–40, 8–40, and 10–40) were formulated
as described above with OH-RNA labeled with AlexaFluor 647 (Integrated
DNA Technologies) at an N:P ratio of 20:1. Formulated fluorescent
OH-RNA/NPs were then supplemented with 10% additional PBS or to a
final concentration of 10% fetal bovine serum (FBS; Gibco) and incubated
at 37 °C for 0, 2, or 6 h prior to analysis by agarose gel (4%)
electrophoresis (1 μg RNA per lane). For the PBS group and t = 0 time point, the NPs and the respective diluent were
warmed to 37 °C and mixed immediately before loading. The gel
was subsequently imaged on a LICOR Odyssey imaging system. To evaluate
the effect of serum incubation on complex activity, lead carriers
were formulated with 3pRNA, diluted in 80% FBS for 0, 2, 6, 12, 18,
or 24 h, and activity evaluated in A549-Dual reporter cells (Invivogen)
as described above at a concentration of 2 nM RNA. Data are plotted
as a percentage of luminescence at t = 0 h after
subtracting background signal from vehicle (PBS) treated controls.
complex stability in serum by agarose gel electrophoresis, lead carriers
(4–50, 6–40, 8–40, and 10–40) were formulated
as described above with OH-RNA labeled with AlexaFluor 647 (Integrated
DNA Technologies) at an N:P ratio of 20:1. Formulated fluorescent
OH-RNA/NPs were then supplemented with 10% additional PBS or to a
final concentration of 10% fetal bovine serum (FBS; Gibco) and incubated
at 37 °C for 0, 2, or 6 h prior to analysis by agarose gel (4%)
electrophoresis (1 μg RNA per lane). For the PBS group and t = 0 time point, the NPs and the respective diluent were
warmed to 37 °C and mixed immediately before loading. The gel
was subsequently imaged on a LICOR Odyssey imaging system. To evaluate
the effect of serum incubation on complex activity, lead carriers
were formulated with 3pRNA, diluted in 80% FBS for 0, 2, 6, 12, 18,
or 24 h, and activity evaluated in A549-Dual reporter cells (Invivogen)
as described above at a concentration of 2 nM RNA. Data are plotted
as a percentage of luminescence at t = 0 h after
subtracting background signal from vehicle (PBS) treated controls.