Poly(ethylene glycol)-Based Peptidomimetic "PEGtide" of Oligo-Arginine Allows for Efficient siRNA Transfection and Gene Inhibition

Abstract

While a wide range of experimental and commercial transfection reagents are currently available, persistent problems remain regarding their suitability for continued development. These include the transfection efficiency for difficult-to-transfect cell types and the risks of decreased cell viability that may arise from any transfection that does occur. Therefore, research is now turning toward alternative molecules that improve the toxicity profile of the gene delivery vector (GDV), while maintaining the transfection efficiency. Among them, cell-penetrating peptides, such as octa-arginine, have shown significant potential as GDVs. Their pharmacokinetic and pharmacodynamic properties can be enhanced through peptidomimetic conversion, whereby a peptide is modified into a synthetic analogue that mimics its structure and/or function, but whose backbone is not solely based on α-amino acids. Using this technology, novel peptidomimetics were developed by co- and postpolymerization functionalization of substituted ethylene oxides, producing poly(ethylene glycol) (PEG)-based peptidomimetics termed "PEGtides". Specifically, a PEGtide of the poly(α-amino acid) oligo-arginine [poly(glycidylguanidine)] was assessed for its ability to complex and deliver a small interfering ribonucleic acid (siRNA) using a range of cell assays and high-content analysis. PEGtide-siRNA demonstrated significantly increased internalization and gene inhibition over 24 h in Calu-3 pulmonary epithelial cells compared to commercial controls and octa-arginine-treated samples, with no evidence of toxicity. Furthermore, PEGtide-siRNA nanocomplexes can provide significant levels of gene inhibition in "difficult-to-transfect" mouse embryonic hypothalamic (mHypo N41) cells. Overall, the usefulness of this novel PEGtide for gene delivery was clearly demonstrated, establishing it as a promising candidate for continued translational research.

Figure 1

General structures of peptides and side-chain-functionalized poly(ethylene glycol)s as PEG-based peptidomimetics (Rⁿ: variable side chain; In: initiator).

Scheme 1. Synthetic Route to Poly(glycidylguanidine) (4), Starting from (±)-tert-Butyl N-(2-Oxiranylmethyl)carbamate

Figure 2

MALDI-TOF MS spectrum of poly(glycidylguanidine). The molecular weight distributions of 677, 793, 908, 1023, 1138, 1253, 1368, 1483, and 1598 are from the main series, and the molecular weight distributions of 661, 776, 892, 1007, 1122, 1237, 1352, 1467, 1582, and 1697 are from the fragmentation peak series. A version of this spectrum labeled with exact masses is available in the Supporting Information.

Figure 3

(A) Particle size and polydispersity indices and (B) ζ potential of PEGtide–siRNA nanoparticles at N/P ratios of 10–100 [±standard error of the mean (SEM), n = 5].

Figure 4

Quantitative high-content analysis (HCA) of valinomycin and MG132 positive controls and PEGtide–siRNA-nanoparticle-mediated toxicity in Calu-3 cells measured at 24 h post-administration. (A) Cell count, (B) mitochondrial membrane potential (MMP), (C) cytochrome c (Cyt c) release, and (D) plasma membrane permeability (PMP). Y axes represent the fluorescence intensity in arbitrary units (a.u.) (one-way analysis of variance vs untreated cells, n = 3 ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

HCA 20× fused image analysis of PEGtide–siRNA uptake in Calu-3 cells 24 h post-transfection. The cells were treated with 100 nM of FITC-tagged siRNA nanoparticles (green) and were subsequently stained for cell nuclei using Hoechst nuclear stain (blue) and cell membrane using phalloidin–tetramethylrhodamine B isothiocyanate (TRITC) (red). (A) Untreated cells, (B) RNAiFect, (C) PEGtide N/P = 9, (D) expanded view of PEGtide N/P = 15, and (E) quantitative HCA analysis of RNAiFect vs PEGtide–siRNA nanoparticle uptake in Calu-3 cells measured at 2, 4, and 24 h post-administration (two-way ANOVA, n = 3 ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

(A) Percentage of luciferase knockdown in Calu-3 cells after treatment with PEGtide–siRNA or octa-arginine siRNA nanoparticles vs nontargeting controls using 100 nM of antiluciferase siRNA 24 h post-transfection (two-way ANOVA, n = 3 ± SEM, ^†p < 0.05 ^††p < 0.01, ^† vs RNAiFect, * vs octa-arginine). (B) Percentage of luciferase knockdown in difficult-to-transfect N41 neuronal cells demonstrated significant levels of knockdown that were comparable to those in commercial controls (one-way ANOVA, n = 3 ± SEM, * p < 0.05, ** p < 0.01, significance vs nontargeting controls).

Figure 7

Proposed structure of the products formed under MALDI conditions for the main series of the molecular weight distribution in MS analysis.