Figure Legends
Figure 1. Scheme of intracellular delivery using siRNA/fusion peptide complexes. The complex could be self-assembled via electrostatic interaction between negatively charged siRNA and positively charged arginine-rich region of fusion peptides as well as hydrophobic interaction between charge-shielded siRNA and hydrophobic SPACE region of fusion peptides. The complex penetrated cellular membrane via endocytosis pathway. siRNA escaped from endosomes knocked down the specific gene through RISC formation.
Figure 2. Characterization of siRNA/peptide self-assembled complexes. (A) Complex formation was checked using a gel retardation assay. The 21-nt siRNA was mixed with each peptide: SPACE, R11, TAT, S-R7, S-R11, and S-R15 at N/P ratios of 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, and 100:1. After a 30-minute incubation, complexes mixed with a 6X loading dye were loaded into 2% (w/v) agarose gel stained with TopRed. Gel electrophoresis was run in TAE buffer at 25 VA for 30 minutes. The gel was visualized through a ChemiDoc™ XRS+ System. The brightness and contrast of each picture were adjusted. (B) Size and zeta potential of complexes were measured using dynamic light scattering. The 21-nt siRNA of 200 nM final concentration was incubated for 30 minutes with each peptide: SPACE, R11, S-R7, S-R11, and S-R15. After filtration and vortexing, each complex was loaded in the cell and analyzed through Nano ZS. Bars represented the average ± standard deviation. P-value was calculated using a t-test compared to that of SPACE (**; p<0.01. independent repeat=3). (C) siRNA stability was tested in serum. In the left pictures, each 21-nt siRNA/peptide complex was incubated in 10% FBS for 24, 48, 72, and 96 hours. In the right pictures, each complex was incubated in 50% FBS for 4, 8, 12, 24, and 48 hours. Then, samples mixed with a 6X loading dye were loaded into 2% (w/v) agarose gel stained with TopRed. Gel electrophoresis was run in TAE buffer at 25 VA for 25-30 minutes. The gel was visualized through a gel documentation system. The brightness and contrast of each picture were adjusted.
Figure 3. Cellular uptake evaluation of siRNA/peptide complexes into (A) HeLa, (B) HDFn, and (C) HaCaT cells using flow cytometry. 265 μL complex of the final 200 nM Cy3-labeled IL10-siRNAs was delivered into each 3.0×105 cells in a 6-well plate via PC: Lipofectamine™ 2000 as a commercialized positive control, SPACE, R11, S-R7, S-R11, and S-R15 (20:1 N/P ratio) for 4 hours. Fluorescent cells of siRNA only and each condition exhibited in red and green populations, respectively. The population of fluorescent-positive cells was expressed as a percentage.
Figure 4. Cellular uptake evaluation of siRNA/peptide complexes into HeLa cells using fluorescence and confocal microscopes. (A) Cellular uptake of Cy3-labeled IL10-siRNA/peptide complexes using a fluorescence microscope. 132 μL complex of the final 200 nM Cy3-labeled IL10-siRNAs were delivered into each 1.0×105 HeLa cells in a 24-well plate via PC: Lipofectamine™ 2000 as a commercialized positive control, SPACE, R11, S-R7, S-R11, and S-R15 (20:1 N/P ratio) for 4 hours. Nucleus and actin filaments were labeled using Hoechst 33342 (blue) and Phalloidin (green), respectively. The complex was observed in orange color at 200× magnification (scale bar=100 μm). (B) Single-molecule images of siRNA/S-R15 complex using a super-resolution radial fluctuation. 2.0×104 cells of HeLa were incubated in a 35 mm confocal dish. 20 μL complex of the final 50 nM of siRNA and S-R15 (20:1 N/P ratio) was applied to the cells for 4 hours. Cy3-labeled IL10-siRNA and FITC-labeled S-R15 peptide were observed in magenta and green, respectively, at 900× magnification (scale bar=1 μm). Actin filaments were labeled using a SiR-actin kit (red). Fluorescence images of Cy3, FITC, and SiR-actin were merged using Image J software. Co-localization of the complex was visualized with arrowed white spot-like areas in a merged image (scale bar=10 μm). (C) Cellular internalization of siRNA/S-R15 complex using a Z-stack image. Actin filaments were labeled using a SiR-actin kit (red). The arrowed white spot-like areas demonstrated co-localization of siRNA and peptide in the cytoplasm at 900× magnification (scale bar=5 μm). Right, and bottom images showed a cross-sectional z-axis image of the arrowed white spot.
Figure 5. Target GAPDH mRNA knockdown in HeLa and HaCaT by siRNA/peptide complexes using quantitative RT-PCR. 132 μL complex of the final 200 nM GAPDH-siRNAs was delivered into each 1.0×105 HeLa cells in a 24-well plate using Lipofectamine™ 2000 as a commercialized positive control, SPACE, R11, S-R7, S-R11, and S-R15 (20:1 N/P ratio) for 5 hours. 100 ng of total RNAs isolated from cells was reverse-transcribed into cDNA. 10 ng cDNA was used for PCR reaction with GAPDH-specific forward and reverse primers. Relative mRNA expression levels were calculated using the ΔΔCt method based on the housekeeping β-actin expression level. The relative expression levels of GAPDH mRNA were normalized by mRNA expression of siRNA only. The data represented mean ± standard deviation (*; p<0.05, **; p<0.01 and independent n≥3).
Figure 6. Endocytosis pathway identification of the siRNA/S-R15 complex into HeLa cells using flow cytometry and various chemical inhibitors. HeLa cells of 4.0×105 were pretreated with each endocytosis inhibitor (10 μg/mL of chlorpromazine, 5 mM of methyl-β-cyclodextrin, 1 μM of cytochalasin D, and 1 μg/mL of filipin III) for 30 minutes. 150 μL complex of the final 100 nM Cy3-labeled siRNA at 20:1 N/P ratio was delivered into the cells for 4 hours. Each fluorescent cell population was counted using detached cells via FITC (upper row) and Cy3 (lower row) intensities, respectively. Fluorescent cell population w/o and w/ each inhibitor exhibited in red and green, respectively. The reduced population of fluorescent-positive cells was expressed as a percentage.
Figure 7. Cytotoxicity test of three fusion peptides using a lactate dehydrogenase (LDH) assay. Released LDH activities were measured from 8.0×103 HDFn cells under each fusion peptide at different concentrations in a 96-well plate using a cytotoxicity kit. LDH activity of the cells treated with each peptide was normalized with the LDH activity without peptide. The data represented the mean ± standard deviation (independent repeat=3). P-value was calculated using a t-test.