3.2. In vitro cellular uptake evaluation
Cellular uptake efficiencies of each complex were evaluated using flow
cytometry and HeLa, HDFn, and HaCaT cells (Fig. 3). Using Cy3-labeled
IL10-siRNA, fluorescent cells with siRNA only and each condition
exhibited in red and green populations, respectively. The percentage
represented the population of fluorescent-positive cells over total
cells. Complexes using S-R7, S-R11, and S-R15 showed the high cellular
uptake efficiencies of 95.59%, 85.24%, and 78.18% in HeLa cells (Fig.
3A), 99.75, 99.79 and 99.55% in HDFn cells (Fig. 3B), and 95.46%,
79.07% and 99.99% in HaCaT cells (Fig. 3C), respectively. These
efficiencies were higher than 71.34% in HeLa cells, 86.95% in HDFn
cells, and 79.79% in HaCaT cells treated by Lipofectamine™ 2000 as a
commercialized positive control. The complex using R11, a single
peptide, showed cellular uptake efficiency of 92.58% in HeLa cells,
88.48% in HDFn cells, and 99.82% in HaCaT cells. In contrast, the
complex using SPACE showed negligible cellular uptake efficiency in HeLa
cells (4.51%), HDFn cells (0.25%), and HaCaT cells (0.19%).
Cellular uptake of each complex was observed using a fluorescence
microscope and HeLa cells (Fig. 4A). Images represented Cy3-labeled
siRNA of orange fluorescence, the nucleus of blue fluorescence, and
actin filament of green fluorescence. Orange fluorescence inside of
cells was observed in the images of Lipofectamine™ 2000 (the second
row), R11 (the fourth row), S-R7 (the fifth row), S-R11 (the sixth row),
and S-R15 (the seventh row) in Fig. 4A. Complexes using fusion peptides
showed orange spots in the cytosol. On the other hand, Lipofectamine™
2000 showed dispersed orange fluorescence within the cytosol, and an R11
complex showed orange fluorescence spots and spread within the cytosol.
In contrast, siRNA only and SPACE complex did not show any orange
fluorescence in the first and third rows of Fig. 4A.
In addition, co-localization and cellular internalization of the
siRNA/peptide complex were confirmed using a confocal microscope with
super-resolution at the single-molecule level (Fig. 4B&C). The confocal
images represented Cy3-modified siRNA of magenta fluorescence and
FITC-modified S-R15 peptide of green fluorescence (Fig. 4B). The arrowed
spot of the merged image indicated the co-localization of siRNA and
S-R15 peptide complex with white fluorescence. The Z-stack image of
confocal microscopy represented intracellular localization of
Cy3-labeled IL10-siRNA and FITC-labeled S-R15 complex in HeLa cells
(Fig. 4C). The arrowed spot of white fluorescence was located in the
cytoplasm around the nucleus.
siRNA delivered by complex knocked down GAPDH mRNA expression in HeLa
and HaCaT cells using quantitative RT-PCR analysis (Fig. 5). In the HeLa
cells, complex using S-R15 reduced relative GAPDH mRNA expression of
38.7% compared to that of siRNA only. This knockdown percentage was
significantly different from that of SPACE (p -value = 0.011) and
comparable to 35.3% using Lipofectamine™ 2000. Complexes using R11,
S-R7, and S-R11 knocked down GAPDH mRNA expression to 52.6, 56.8, and
51.3%, respectively. Moreover, the complex using SPACE induced the
least knockdown of 72.8%. In the HaCaT cells, the 49.8% knockdown
using S-R15 was comparable to 40.8% of Lipofectamine™ 2000 without a
statistically significant difference. These results indicated that these
complexes could knock down mRNA expression in different cell types,
including cancer cell and skin keratinocyte.