Evaluation of PF14-derived cell-penetrating peptides for functional delivery of plasmid DNA

Abstract

Plasmid DNA (pDNA) represents a promising platform for gene therapy due to its substantial coding capacity and capability to support sustained gene expression. However, its intracellular delivery remains a significant challenge attributed to its large molecular size, negative charge, and multiple biological barriers. Cell-penetrating peptides (CPPs) serve as promising non-viral delivery vectors, adept at forming nanoscale complexes that facilitate the intracellular transport of nucleic acids. Among the class of amphipathic CPPs, PepFect14 (PF14) has exhibited remarkable efficacy in nucleic acid delivery, characterized by efficient nanoparticle (NP) formation and cellular uptake. Based on the success of PF14, a series of structural modifications has been systematically investigated to optimize NP characteristics and improve the delivery efficiency. The study evaluates a series of PF14-derived RNA delivery peptides featuring distinct amino acid substitutions and hydrophobic modifications for their efficacy in pDNA delivery. The CPP–pDNA NPs were characterized for complexation efficiency, physicochemical properties, stability, and post-transfection functional delivery and cell viability. The functional delivery of a luciferase-encoding plasmid was assessed in CHO-K1 cells, as well as heparan sulfate-deficient CHO 2242 cells, to evaluate how peptide modifications impact delivery performance and to elucidate the role of cell-surface heparan sulfate in CPP-mediated pDNA delivery. Most peptides efficiently condensed pDNA and formed nanoscale NPs with generally favorable physicochemical characteristics. Notably, peptides with histidine substitutions or specific hydrophobic modifications demonstrated enhanced functional delivery, while the performance of other hydrophobic variants displayed variability, underscoring the necessity for a careful balance of hydrophobicity to optimize NP formation, stability, and intracellular release. Reduced transfection efficiency in CHO 2242 cells further emphasized the critical role of cell-surface heparan sulfate in the functional delivery of pDNA. Overall, the findings of this study elucidate the significant influence of structural modifications on the performance of PF14-derived CPPs and contribute to the rational design of advanced CPP-based non-viral gene delivery systems.