Optimizing nucleic acid delivery using PF14 peptide and lipid nanoparticle systems

Nucleic acid-based therapeutics are a rapidly expanding class of precision medicines capable of directly modulating gene expression. However, their clinical application is limited by challenges in cellular delivery, including large molecular size, hydrophilicity, and susceptibility to enzymatic degradation. To address these barriers, advanced delivery platforms such as cell-penetrating peptides and lipid nanoparticles (LNPs) have been developed. This study evaluates two delivery systems: the cell-penetrating peptide PepFect-14 (PF14) and LNP formulations, for enhancing nucleic acid delivery and cellular uptake. PF14 facilitates intracellular transport through peptide-nucleic acid complex formation, while LNPs protect cargo from degradation and can promote endosomal escape. Their performance was assessed under different conditions using luciferase-based reporter systems in HeLa cells. PF14-mediated delivery of the splice-switching oligonucleotide ON-705 in HeLa 705 cells showed efficient splice correction, with activity increasing in a dose- and molar ratio- dependent manner, reaching a plateau at a 1:10 ratio. Delivery efficiency was significantly influenced by formulation conditions, with Opti-MEM outperforming standard media and sugar-based buffers. Polymer excipients also affected activity as PVA18, PVA40, and PVP40 enhanced performance, while low molecular weight PVP reduced efficacy. In parallel, LNP-mediated delivery of luciferase mRNA in wild-type HeLa cells demonstrated robust, dose-dependent protein expression. Both systems maintained high cell viability (80-100%). Overall, these findings highlight the importance of formulation optimization in improving nucleic acid delivery and provide practical insights for enhancing peptide- and lipid-based therapeutic platforms.