Supplementation with ions enhances the efficiency of nucleic acid delivery with cell-penetrating peptides

The successful delivery of therapeutic nucleic acids (NAs) into eukaryotic cells is essential for numerous biomedical applications, including gene therapy, gene silencing, and genome editing. Cell-penetrating peptides (CPPs) have claimed significant attention as delivery vehicles due to their inherent ability to penetrate cellular membranes and efficiently transport cargo, including NAs, into the cells. However, further optimization and a deeper understanding of underlying mechanisms are necessary for such transfection methods. Previous studies have demonstrated that Ca²⁺ ions can significantly enhance NA delivery efficiency when included in transfection media or CPP/NA nanoparticles during preparation. Similar effects have been observed for Mg²⁺, but the impact of other ions in this context has not been thoroughly investigated. In this study, we supplemented the CPP/NA formulations with various inorganic biocompatible ions by introducing solutions of the respective salts to colloidal nanoparticles at the preparation stage. Our results indicated that supplementing the CPP/NA formulations with certain salt solutions enhanced the biological effect achieved with NAs while also influencing nanoparticle size, surface charge, complexation stability, and, to some extent, the internalization route. Our findings offer valuable insights for optimizing the formation of CPP nanoparticles to improve NA delivery efficiency.