Mechanisms of nanoparticle-mediated siRNA transfection by melittin-derived peptides

Abstract

Traditional peptide-mediated siRNA transfection via peptide transduction domains exhibits limited cytoplasmic delivery of siRNA due to endosomal entrapment. This work overcomes these limitations with the use of membrane-destabilizing peptides derived from melittin for the knockdown of NFkB signaling in a model of adult T-cell leukemia/lymphoma. While the mechanism of siRNA delivery into the cytoplasmic compartment by peptide transduction domains has not been well studied, our analysis of melittin derivatives indicates that concurrent nanocomplex disassembly and peptide-mediated endosomolysis are crucial to siRNA transfection. Importantly, in the case of the most active derivative, p5RHH, this process is initiated by acidic pH, indicating that endosomal acidification after macropinocytosis can trigger siRNA release into the cytoplasm. These data provide general principles regarding nanocomplex response to endocytosis, which may guide the development of peptide/siRNA nanocomplex-based transfection.

Figure 1

(a) Scheme for formulation of albumin-stabilized p5RHH/siRNA nanoparticles. (b) Wet-mode AFM imaging of p5RHH/siRNA nanoparticles reveals an average particle size of ~55 ± 18 nm (n = 128).

Figure 2

(a–d) 40 minute uptake of p5RHH/Alexa488-siRNA nanoparticles shows that 60% of the treated cells take up (a) p5RHH/siRNA nanoparticles. The presence of endocytosis inhibitors indicates that (b) 100μg/mL filipin (caveolae inhibitor) and (c) 10μM PAO (clathrin mediated endocytosis inhibitor) do not inhibit p5RHH/siRNA nanoparticle uptake. Alternatively, treatment with (d) macropinocytosis inhibitor (EIPA, 80μM) nearly abolishes nanoparticle uptake. (e–j) Colocalization as determined by confocal microscopy shows that p5RHH/Cy-3 siRNA nanoparticles are taken up with FITC-70kDa dextran (j) but not FITC-transferrin (i). Scale bar 10μm.

Figure 3

(a–c) Bafilomycin A1 does not inhibit uptake of p5RHH/Alexa488-siRNA nanoparticles (c) compared to transfection in the absence of bafilomycin A1 (b). (d–f) On the other hand, bafilomycin A1 blocks knockdown of GFP (f) compared to transfection in the absence of bafilomycin A1 (e) indicating that endosomal acidification is crucial for p5RHH mediated siRNA transfection.

Figure 4

(a) Fluorescence from TOPRO3 binding to siRNA increases dramatically at pH ≤ 5.5 when packaged via p5RHH ( ), but not the non-functioning peptide p5RWR ( ). (b) Polyacrylamide gel electrophoresis confirms that p5RHH releases siRNA at pH 4.5 but p5RWR shows no pH-dependent release. (c) p5RHH is also released at low pH with an increase in p5RHH release at pH ≤ 5.5. (d) Freed p5RHH is capable of hemolysis, leading to increased hemoglobin release at pH ≤ 5.5. (e–h) Acridine orange release assays show that p5RHH/siRNA nanoparticles are able to disrupt endosomes (h) when tested in tissue culture, as exhibited by dye release similar to that of 100μM chloroquine (f), whereas p5RWR cannot (g). Scale bar 50μm.

Figure 5

(a) Knockdown of GFP in B16 GFP cells reveals that only p5RHH can successfully deliver GFP siRNA to the cytoplasm, whereas p5RWR can not even with endosomal escape induced by chloroquine. (b) Flow cytometry reveals both p5RWR and p5RHH deliver similar amounts of alexa 488-labeled siRNA. Untreated control ( ); 50nM a488 siRNA/p5RWR ( ); 50nM a88 siRNA/p5RWR + chloroquine ( ); 50nM a488 siRNA/p5RHH ( ); 50nM a88 siRNA/p5RHH + chloroquine ( ). Confocal microscopy (scale bar 10μm) reveals that p5RWR (c) delivers siRNA by remains in punctate vesicles whereas p5RHH achieves cytoplasmic distribution (d). Simultaneous incubation with chloroquine is required to release siRNA to the cytoplasm when transfected by p5RWR (e) but has no effect on p5RHH-mediated transfection (f).

Figure 6

(a,b) Western blotting demonstrates a dose-dependent decrease in p100/p52 or p65 expression that is not seen when treating F8 cells with scrambled siRNA. (c) Alamar blue assays 48 hours post transfection reveals that scrambled siRNA (■) does not affect F8 cell viability. Knockdown of the canonical NFkB pathway with p65 siRNA (▲) has an IC50 of nearly 200nM. Targeting the non-canonical NFkB pathway with p100/p52 siRNA (●) yields an IC50 of 100nM. However, a nanoparticle formulation simultaneously carrying siRNA to block both canonical and non-canonical NFkB pathways (◆) improves the IC50 to 50nM. IVIS imaging (scale bar 5mm) reveals tumor localization of Cy5.5 labeled siRNA to the tumor of treated mice (e), and is confirmed by confocal microscopy (g) (scale bar 50μm). Non-treated controls shown for comparison (d, f)