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.