Cell-Penetrating Peptide Nanoparticles for Delivery of Splicing-Correction Oligonucleotides and DNA Origami

Abstract

Cell-penetrating peptides (CPPs) are emerging as versatile, non-viral vehicles for nucleic-acid therapeutics, yet their cargo range and in-cell efficiency remain limited. In particular, most studies have not compared how the same CPP sequence handles cargos of very different size and architecture; moreover, there have been no attempts yet to deliver the DNA origami using the CPPs. Here, we asked how the three PepFect14-derived CPPs — PF14, Peptide 1, and PF14-Lys-Ile9 — perform with small versus bulky nucleic acids. Nanocomplexes were prepared by simple aqueous mixing and characterised by DLS/TEM, giving uniform spheroidal particles 100–160 nm in diameter (PDI ≤ 0.30). Functional delivery was then benchmarked for (i) a splice-correction oligonucleotide (SCO-705), (ii) a fluorescent three-arm DNA-origami probe (Nano-Cy3), and (iii) a luciferase-silencing siRNA embedded in, or tethered to, the same origami scaffold. In HeLa pLuc705 cells, Peptide 1 restored luciferase ~34-fold, exceeding PF14 (24-fold) and PF14-Lys-Ile9 (21-fold), while flow cytometry showed PF14-Lys-Ile9 achieved the highest Nano-Cy3 uptake in U87 cells without a corresponding gain in splice rescue, pinpointing endosomal escape as the bottleneck. With siRNA packed into the native LUC nanohydrogel, PF14-Lys-Ile9 drove the deepest knock-down (~72 %), whereas activity halved when siRNA was supplied as an isolated linker duplex. Overall, Peptide 1 is best suited to delivering small oligonucleotides, whereas PF14-Lys-Ile9 is more effective for larger, origami-based constructs — insights that chart distinct optimisation routes for future CPP nanocarriers.