Enhancing the Membrane Destabilization Capacity of DNA–Surfactant Conjugates for Improved Cytosolic Delivery of Nucleic Acids

Enhancing the endosomal escape of nucleic acids remains a critical bottleneck in improving their therapeutic efficacy. Inspired by advancements in lipid nanoparticles, we describe a chemical approach to deliver multi-tailed DNA-surfactant conjugates (DSCs) into cells using nucleic acid nanocapsules (NANs). NANs are enzyme degradable, crosslinked DNA micelles that exhibit enhanced membrane destabilizing properties. Previously, we reported the successful delivery of therapeutic oligonucleotides to cells using single-tailed DSCs delivered by esterase-cleavable NANs. This work features the rational design of a set of asymmetrically synthesized hydrophobic micelle crosslinkers using a synthetic approach that incorporates either one or two alkyl chains and an esterase-labile group into a nonionic surfactant flanked by clickable azides. These crosslinkers are covalently incorporated into the NAN construct, and upon their enzymatic degradation within the cell, multi-tailed DSCs are released. We show that these DSCs have superior transfection capabilities over single-tailed DSCs. Additionally, we show that these multi-tailed DSCs improve DNAzyme-mediated gene silencing and enhance mRNA delivery in vitro, owing to their enhanced membrane disruption capabilities reflected in both cell studies and molecular dynamics simulations. Importantly, our chemical approach enables the formation of multi-tailed surfactants in situ, post micelle assembly. This allows an oligonucleotide nanomaterial delivery platform to act as a "pro-drug" where the active therapeutic and the potential for its cytosolic delivery are the product of the nanoformulation's degradation.