Enhancing intracellular delivery of nucleic acid cargo by cell-penetrating peptide-based nanoparticles with small-molecule additives by post-formulation incorporation

: Nucleic acid-based (NA) therapeutics offer versatile strategies for treating genetic and acquired disorders; however, their clinical application is hindered by inefficient intracellular delivery and degradation within endo-lysosomal compartments. Cell-penetrating peptides (CPPs), such as PepFect14 (PF14), represent a promising class of non-viral vectors for NA delivery. In this study, we systematically evaluated a panel of small-molecule additives to optimize the entry and intracellular trafficking of PF14-NA nanoparticles (NP). These additives target key cellular processes, including uptake enhancement, endosomal escape, exocytosis inhibition, and modulation of autophagy, to improve the performance of PF14-NA NPs. Using splice-correction oligonucleotides (SCOs), siRNA, mRNA, and plasmid DNA as model cargos, we demonstrate that the application of endosomal escape enhancers and autophagy modulators significantly enhanced the functional delivery of NA without introducing cytotoxicity. Confocal microscopy and flow cytometry analyses reveal that the increased efficacy primarily stems from improved endo-lysosomal release, increased by endosomal escape enhancers. Importantly, incorporating the additives into PF14-NA NPs yields a stronger effect compared to their supplementation in the culture medium. These findings highlight the potential of the small-molecule-assisted CPP-based delivery system to overcome endosomal barriers, thereby enhancing the cytosolic bioavailability of NA. The approach can offer a strategy to advance the efficiency of non-viral gene delivery platforms.