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.