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.