3.1. Confirmation and characterization of siRNA/peptide
complexes
The formation of siRNA/peptide complexes at different N/P ratios was
confirmed by electrophoretic mobility shift (Fig. 2A). As a result, R11,
S-R7, S-R11, and S-R15 retarded the siRNA band. As fusion peptides,
S-R7, S-R11, and S-R15 showed complete retardation of siRNA at ratios
over 20:1, 10:1, and 40:1, respectively. Partial retardation was
observed with R11, a single peptide, based on the blur siRNA band at
ratios over 30:1. On the other hand, SPACE and TAT did not retard the
siRNA band at all for all N/P ratios. Because TAT, a single peptide, did
not form a condensed complex with siRNA, other peptides except for TAT
peptide were used for the following experiments.
The size and zeta potential of the complex were measured three times
using dynamic light scattering (Fig. 2B). Complexes using SPACE, R11,
S-R7, S-R11, and S-R15 had average sizes of 647.7, 413.7, 327.1, 457.0,
and 287.0 nm, respectively. Complexes using SPACE, R11, S-R7, S-R11, and
S-R15 had average polydispersity indexes (PdI) of 0.89, 0.23, 0.22,
0.23, and 0.04, respectively. Complexes using SPACE, R11, S-R7, S-R11,
and S-R15 had average zeta potentials of -28.33, -11.77, 5.23, 6.00, and
6.08, respectively.
The stability of siRNA under complexes was assessed using serum
incubation (Fig. 2C). First, each complex was incubated with 10% FBS to
simulate a cell culture condition. Interestingly, complexes using S-R15
and S-R11 maintained the siRNA stability until 96 hours. On the other
hand, siRNA bands of other complexes gradually disappeared over time.
Decomposition rates in 10% FBS were S-R15, S-R11, R11, siRNA only,
S-R7, and SPACE in increasing order. Second, each complex was incubated
with 50% FBS to simulate extreme decomposition conditions.
Interestingly, the S-R15 complex only maintained siRNA stability until
48 hours. On the other hand, siRNA bands of other complexes gradually
disappeared and completely disappeared at 24 hours. Decomposition rates
in 50% FBS were S-R15, S-R11, R11, S-R7, siRNA only, and SPACE in
increasing order. In summary, S-R15 showed the best siRNA stability
under complex in both conditions.