Comparison of Cell-Penetrating and Fusogenic TAT-HA2 Peptide Performance in Peptideplex, Multicomponent, and Conjugate siRNA Delivery Systems

Abstract

In this study, the performance of the cell-penetrating and fusogenic peptide, TAT-HA2, which consists of a cell-permeable HIV trans-activator of transcription (TAT) protein transduction domain and a pH-responsive influenza A virus hemagglutinin protein (HA2) domain, was comparatively evaluated for the first time in peptideplex, multicomponent, and conjugate siRNA delivery systems. TAT-HA2 in all three systems protected siRNA from degradation, except in the conjugate system with a low Peptide/siRNA ratio. The synergistic effect of different peptide domains enhanced the transfection efficiency of multicomponent and conjugate systems compared to that of peptideplexes, which was attributed to the surface configuration of TAT-HA2 peptides depending on the nature of attachment. Particularly, the multicomponent system showed better cellular uptake and endosomal escape than the peptideplexes, resulting in enhanced siRNA delivery in the cytoplasm. In addition, the presence of cleavable disulfide bonds in multicomponent and conjugate systems promoted the effective siRNA delivery in the cytoplasm, resulting in improved gene silencing activity. The multicomponent system reduced the level of luciferase expression in SKOV3 cells to 45% (±4). In contrast, the conjugate system and the commercially available siRNA transfection agent, Lipofectamine RNAiMax, caused luciferase suppression down to 55% (±2) at a siRNA dose of 100 nM. For the same dose, the peptideplex system could only reduce the luciferase expression to 65% (±5). None of the developed systems showed significant toxicity at any dose. Overall, the TAT-HA2 peptide is promising as a siRNA delivery vector; however, its performance depends on the nature of attachment and, as a result, its surface configuration on the developed delivery system.

Scheme 1. Schematic Representation of the Developed Systems

(A) Peptideplexes. (B) Gold nanoparticle-based multicomponent system (MCS). (C) Conjugate system.

Figure 1

(A) Gel electrophoresis of siRNA/Peptide complexes formed at various Peptide/siRNA ratios: 2.5, 5, 7.5, 10, and 12.5. Control: naked siRNA. (B) Serum stability of the siRNA/Peptide complexes formed at Peptide/siRNA ratios of 5, 7.5, and 10. Serum content: 50% (v/v). Incubation: 6 h at 37 °C. (C) RNAse-exposed siRNA/Peptide complexes at various Peptide/siRNA ratios: 5, 7.5, 10. Control: naked siRNA and RNase exposed naked siRNA. RNase concentration: 0.25% (v/v). Incubation: 6 h at 37 °C. The size (D) and zeta potential (E) values of siRNA/Peptide peptideplexe systems (Peptide/siRNA ratio: 5, 7.5, and 10) measured immediately in PBS or after 24-h incubation in a serum-containing DMEM growth medium. * represents a statistically significant difference (p < 0.05).

Figure 2

(A) The cellular accumulation of peptideplexes at different Peptide/siRNA ratios. Incubation time: 24 h. (B) Time-dependent cellular uptake of siRNA/Peptide peptideplexes (Peptide/siRNA ratio: 10). Applied dose: 100 nM based on siRNA. Incubation times: 15, 30, and 60 min. * represents a statistically significant difference (p < 0.05). (C) Merged fluorescence images of siRNA/Peptide complexes prepared with a Peptide/siRNA ratio of 10. Green: peptideplexes stained by FITC, Red: Lysosome stained by LysotrackerRed, Blue: Nucleus stained by DAPI. Incubation time: 24 h.

Figure 3

(A) Luciferase expression suppression and (B) cell viability of the prepared siRNA/Peptide complexes administered to SKOV3 cells. Peptide/siRNA: 5, 7.5, and 10. Applied siRNA dose: 100, 200, and 400 nM. Commercial Lipofectamine RNAiMax siRNA transfection reagent with a 100 nM siRNA dose. Initial SKOV3 cell density: 1 × 10⁴ cells/well. Incubation time: 24 h. * represents a statistically significant difference (p < 0.05) between the selected cases.

Figure 4

UV–vis spectra of AuNP, PEG-modified AuNP, SPDP, and siRNA-modified AuNP-PEG, and peptide-coated AuNP-siRNA multilayer system (AuNP/Peptide ratio: 1/500 or Peptide/siRNA ratio: 5 is shown here since all other ratios showed a similar trend).

Figure 5

(A) Agarose gel electrophoresis of the AuNP-siRNA-Peptide MCS and naked siRNA. RNase and serum stabilities of the MCSs were prepared at AuNP/Peptide ratios of 1/125, 1/250 and 1/500 (Peptide/siRNA ratios of 1.25, 2.5, and 5, respectively) Serum content: 50% (v/v). Incubation: 6 h at 37 °C. RNase concentration: 0.25% (v/v). Incubation: 1 h at 37 °C. The change in the size (B) and zeta potential (C) values of AuNP-siRNA-Peptide MCSs (AuNP/Peptide ratios: 1/125, 1/250, and 1/500 or Peptide/siRNA ratios: 1.25, 2.5, and 5) after preparation in PBS buffer (PBS) and after 24 h of incubation at 37 °C in serum-containing DMEM growth medium (DMEM). * represents a statistically significant difference (p < 0.05).

Figure 6

(A) The cellular accumulation of AuNP-based MCSs in SKOV3 cells measured by ICP-MS. Applied dose based on siRNA amount: 100 nM. Incubation time: 24 h. (B) Time-dependent cellular uptake of AuNP-siRNA-Peptide MCSs (AuNP/Peptide ratio: 1/500 or Peptide/siRNA ratio: 5). Applied dose: 100 nM based on siRNA. Time: 15, 30, and 60 min. * represents a statistically significant difference (p < 0.05). (C) Fluorescence images of AuNP-siRNA-Peptide MCSs prepared with a AuNP/Peptide ratio of 1/500 (Peptide/siRNA ratio of 5). Applied dose: 100 nM based on siRNA. Green: MCSs stained by FITC, Red: Lysosome stained by LysotrackerRed, Blue: Nucleus stained by DAPI.

Figure 7

(A) Luciferase suppression and (B) cell viability of prepared AuNP-siRNA-Peptide MCSs (AuNP/Peptide ratio: 1/125, 1/250, and 1/500 or Peptide/siRNA ratio: 1.25, 2.5, and 5, respectively). Applied siRNA dose: 100, 50, 25 nM/well. Commercial Lipofectamine RNAiMax siRNA transfection reagent with 100 nM siRNA dose. Initial SKOV3 cell density: 1 × 10⁴ cells/well. Incubation time: 24 h. * represents a statistically significant difference (p < 0.05) between the selected cases.

Figure 8

(A) Gel electrophoresis of Peptide–siRNA conjugates at various Peptide/siRNA ratios: 5, 7.5, and 10. Control: Naked siRNA. (B) Serum stability of the Peptide–siRNA conjugates at Peptide/siRNA ratios of 5, 7.5, and 10. Serum content: 50% (v/v). Incubation: 6 h at 37 °C. (C) RNase-exposed Peptide–siRNA conjugates at various Peptide/siRNA ratios: 5, 7.5, and 10. Control: naked siRNA and RNase-exposed naked siRNA. RNase concentration: 0.25% (v/v). Incubation: 1 h at 37 °C. The change in the size (D) and zeta potential (E) values of Peptide–siRNA conjugate systems (Peptide/siRNA ratios: 5, 7.5, and 10) after preparation in PBS buffer (PBS) and after 24 h of incubation at 37 °C in serum-containing DMEM growth medium (DMEM). * represents a statistically significant difference (p < 0.05) between the selected cases.

Figure 9

(A) Luciferase expression suppression and (B) cell viability of prepared Peptide–siRNA conjugates administered to SKOV3 cells. Peptide/siRNA ratios: 5, 7.5, and 10. Applied siRNA doses: 200, 100, and 50 nM. Commercial Lipofectamine RNAiMax siRNA transfection reagent with a 100 nM siRNA dose. Initial SKOV3 cell density: 1 × 10⁴ cells/well. Incubation time: 24 h. * represents a statistically significant difference (p < 0.05) for selected cases.

Figure 10

(A) Luciferase expression suppression and (B) cell viability of peptideplex, multicomponent, and conjugate systems administered to SKOV3 cells. The siRNA dose is 100 nM for all the systems. The positive control is the commercial Lipofectamine RNAiMax siRNA transfection reagent with a 100 nM siRNA dose. The negative control is the commercial Lipofectamine RNAiMax transfection reagent with scrambled siRNA at a 100 nM siRNA dose. Initial SKOV3 cell density: 1 × 10⁴ cells/well. Incubation time: 24 h. * represents a statistically significant difference (p < 0.05) for selected cases.