Determining the Role of Surfactant on the Cytosolic Delivery of DNA Cross-Linked Micelles

Nucleic Acid Nanocapsules (NANs) are nucleic acid nanostructures that radially display oligonucleotides on the surface of cross-linked surfactant micelles. Their chemical makeup affords the stimuli-responsive release of therapeutically active DNA-surfactant conjugates into the cells. While NANs have so far demonstrated the effective cytosolic delivery of their nucleic acid cargo, as seen indirectly by their gene regulation capabilities, there remain gaps in the molecular understanding of how this process happens. Herein, we examine the enzymatic degradation of NANs and confirm the identity of the DNA-surfactant conjugates formed by using mass spectrometry (MS). With surface enhanced (resonance) Raman spectroscopy (SE(R)RS), we also provide evidence that the energy-independent translocation of such DNA-surfactant conjugates is contingent upon their release from the NAN structure, which, when intact, otherwise buries the hydrophobic surfactant tail in its interior. Such information suggests a critical role of the surfactant in the lipid disruption capability of the DNA surfactant conjugates generated from degradation of the NANs. Using NANs made with different tail lengths (C₁₂ and C₁₀), we show that this mechanism likely holds true despite significant differences in the physical properties (i.e., critical micelle concentration (CMC), surfactants per micelle, <i>N</i>_agg) of the resultant particles (C₁₂ and C₁₀ NANs). While the total cellular uptake efficiencies of C₁₂ and C₁₀ NANs are similar, there were differences observed in their cellular distribution and localized trafficking, even after ensuring that the total concentration of DNA was the same for both particles. Ultimately, C₁₀ NANs appeared less diffuse within cells and colocalized less with lysosomes over time, achieving more significant knockdown of the target gene investigated, suggesting more effective endosomal escape. These differences indicate that the surfactant assembly and disassembly properties, including the number of surfactants per particle and the CMC can have important implications for the cellular delivery efficacy of DNA micelles and surfactant conjugates.