Enhanced nuclear import of DNA-lipid nanoparticles by peptide co-encapsulation

Efficient nuclear delivery of DNA remains a significant challenge for non-viral vectors, limiting their broader application in gene therapy. A key intracellular barrier is represented by the nuclear envelope, where the nuclear pore complex (NPC) restricts passive diffusion of macromolecules, and prevents more than 97% of delivered cytosolic DNA from entering the nucleus. Our study addresses this major limitation with a targeted delivery strategy leveraging nuclear localization signaling (NLS) peptides to enhance nuclear import of plasmid DNA (pDNA). NLS peptides were non-covalently complexed with pDNA and co-encapsulated in lipid nanoparticles (LNPs). Using fluorescently labeled LNPs and pDNA, the intracellular trafficking behavior from cellular uptake to endosomal escape and nuclear entry was characterized with and without the co-encapsulation of the NLS peptide. pDNA-NLS-functionalized LNPs achieved a 2.5-fold increase in transfection efficiency compared to non-complexed controls in vitro. Mechanistic studies indicate that cytosolic importins recognize the co-delivered NLS peptides, facilitating active transport of the released pDNA through the nuclear pore complex. In vivo studies in zebrafish larvae and wildtype mice are consistent with an NLS-dependent enhancement in gene expression, displaying a 10-fold increase after 72 h in mice. These findings demonstrate that LNPs can be combined with NLS peptides to promote non-viral DNA delivery to the nucleus by endogenous nuclear import pathways.