Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

Abstract

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.

Figure 1.

Rational iterative design and general properties of PF6. (a) Chemical structure of the different chemical intermediates of PF6: TP10 backbone (compound 1), succinylated trifluoroquinoline based derivative (compound 2), PepFect3 (compound 3), trifluoroquinoline based derivative conjugated to succinylated Lys7 of TP10 (compound 4), PepFect5 (compound 5) and PepFect6 (compound 6). (b) A representative DLS profile on the distribution of PF6/siRNA particles 30 min after formulation in water and at indicated time points after dilution in 10% serum in optiMEM. (c) Fluorescence microscopy overlay at 1 h after treatment of U2OS cells with 50 nM PF6/Cy5-siRNA. (d) Uptake (RFU) and RNAi response in luc-U2OS cells 24 h after treatment with 50 nM Cy5-labeled luc-siRNA complexed with PF6 or PF3 (left panel). RNAi response after same treatments with- or without co-addition of 100 µM chloroquine (right panel). (e) Liposome leakage at different pH after treatment with 50 nM PF3/siRNA or PF6/siRNA.

Figure 2.

PF6-mediated siRNA delivery into reporter cells. (a) RNAi response in luc-HEK cells at 24 h after treatment with increasing concentration of luc-siRNA using PF6 or LF2000. One hundred nanomolar EGFP siRNA complexed with PF6 (ctrl) and 50 nM naked luc siRNA (Mock) was used as controls. Luminescence (RLU) was normalized to protein content and data presented as expression relative to untreated cells. (b) Dose-response in HepG2 cells after treatment as in (a). (c) Impact of cell confluence. Luc-U2OS cells seeded at indicated densities 1 day prior experiment were treated and analyzed as in (a) using 50 nM siRNA with PF6, LF2000 or RNAiMAX. (d) Flow cytometry histogram analysis of EGFP RNAi response in EGFP-CHO cells at 48 h post treatment with 100 nM free EGFP siRNA (mock) or complexed with LF2000 or PF6. (e) Flow cytometry analysis of EGFP knockdown decay kinetics following single treatment with siRNA. Treatments and subsequent analysis were performed as is in (d) but using 50 nM siRNA. Mock depicts only siRNA and Ctrl corresponds to 50 nM PF6/luc-siRNA. All experiments were performed at least three times in duplicate, showing the standard error of the mean (SEM).

Figure 3.

PF6-mediated siRNA delivery into refractory cells. (a) HPRT1 mRNA knockdown kinetics in Hepa1c1c7 cells following treatment with PF6, LF2000 or RNAiMAX in complex with 50 nM siRNA. Values represent mean normalized to GAPDH mRNA and presented as percent of untreated cells. qPCR analysis of HPRT1 mRNA levels at 24 h post treatment of (b) primary MEF cells, (c) Huvec cells, (d) Jurkat cells or (e) mES-cells with HPRT1-siRNA using PF6, LF2000, RNAiMAX or Transductin as indicated. One hundred nanomolar luc-siRNA complexed with PF6 (Ctrl) and 100 nM naked HPRT1-siRNA (mock) were used as controls. Key applies to b–e.

Figure 4.

PF6/siRNA delivery into mES cells. (a) Fluorescence microscopy analysis of Oct4 expression and genomic staining (DAPI) in mES cells 72 h after treatment with PF6/HPRT1-siRNA, PF6/Oct4-siRNA and Oct4-siRNA only (mock). Ctrl depicts cells without primary antibody. (b) Oct4 RNAi response measured by qPCR 24 h after treatment with 50 nM PF6/Oct4-siRNA or PF6/HPRT1-siRNA. GAPDH was used as internal standard.

Figure 5.

Analysis of the transcriptome and proteome after PF6/siRNA treatment. (a) Full-genome microarray analysis profile after 24 h of HeLa cells treated with PF6 or LF2000 complexed with 50 nM HPRT1-siRNA. Red line indicates a 1.6-fold increase/decrease and blue and red dots indicate genes with significantly altered expression compared to untreated cells. (b) Mass-spectrometry based proteomic analysis of HeLa cells after treatment with 100 nM siRNA in serum-supplemented media either using PF6 or LF2000. Results were normalized to treatments with only siRNA and presented as normal probability plot with a 99% confidence limit. (c) Mass-spectrometry based proteomic analysis of HeLa cells after treatment with 100 nM EGFP-siRNA in serum-supplemented media either using PF6 or RNAiMAX. Results were normalized to treatments with only siRNA and presented as Venn diagram displaying the deregulated proteins with each reagent.

Figure 6.

PF6-mediated siRNA delivery in vivo. (a) Analysis of IL-6 serum levels by ELISA 24 h following i.v. administration of 1 mg/kg of naked siRNA, PF6/siRNA formulated in 5% glucose or glucose only (vehicle). LPS (10 µg) was used as positive control (n = 5 for each group). (b) HPRT1 knockdown in kidney at 72 h post i.v. treatment with 1 mg/kg of PF6/HPRT1-siRNA. Vehicle (5.4% mannitol), naked siRNA (mock) and 1 mg/kg of PF6/luc-siRNA (Ctrl) were used as controls. Organs were lysed, mRNA extracted and HPRT1 mRNA measured by qPCR. These are mean values of HPRT1 normalized to GAPDH mRNA and presented as percent of untreated animals. Error bars indicate SEM, n = 3–6 for each group. (c) HPRT1 RNAi response in indicated organs 72 h after systemic delivery of 1 mg/kg PF6/HPRT1-siRNA formulated in 5.4% mannitol. Organs were lysed, mRNA extracted and HPRT1 mRNA measured by qPCR. These are mean values of HPRT1 normalized to GAPDH mRNA and presented as percent of untreated animals. Error bars indicate SEM, n = 3–6 for each group. (d) Transaminase (ALT/AST) and creatinine serum levels at 72 h after treatment as in (a) (n = 5 for each group) and corresponding histopathology analysis of hematoxylin–eosin stained tissue sections (kidney, lung and liver) from mice treated with 1 mg/kg PF6/siRNA (e). Except for (a), all in vivo formulations were administered in 5.4% mannitol solution.

Figure 7.

Assessment of PF6-mediated siRNA delivery in vivo using a bioluminescence model. (a) In vivo bioluminescence imaging of luciferase expression following i.v. delivery of 1 mg/kg of luc-siRNA, with or without PF6, at Day 3 and 5 post injection in animals with expression of luc from liver. (b) Luc knockdown over time after treatments with 0.2 or 1 mg/kg of PF6/luc-siRNA or 1 mg/kg naked luc-siRNA (mock). (c) Luc knockdown over time after treatments with 1 mg/kg luc-siRNA using hydrodynamic injection (HI) or 1 mg/kg of PF6/luc-siRNA. Naked luc-siRNA (mock) (1 mg/kg) was used as control. At least four animals were included in each treatment group. All in vivo formulations were administered in 5.4% mannitol solution.