Determining\nthe Role of Surfactant on the Cytosolic\nDelivery of DNA Cross-Linked Micelles

Nucleic\nAcid Nanocapsules (NANs) are nucleic acid nanostructures\nthat radially display oligonucleotides on the surface of cross-linked\nsurfactant micelles. Their chemical makeup affords the stimuli-responsive\nrelease of therapeutically active DNA–surfactant conjugates\ninto the cells. While NANs have so far demonstrated the effective\ncytosolic delivery of their nucleic acid cargo, as seen indirectly\nby their gene regulation capabilities, there remain gaps in the molecular\nunderstanding of how this process happens. Herein, we examine the\nenzymatic degradation of NANs and confirm the identity of the DNA–surfactant\nconjugates formed by using mass spectrometry (MS). With surface enhanced\n(resonance) Raman spectroscopy (SE(R)RS), we also provide evidence\nthat the energy-independent translocation of such DNA–surfactant\nconjugates is contingent upon their release from the NAN structure,\nwhich, when intact, otherwise buries the hydrophobic surfactant tail\nin its interior. Such information suggests a critical role of the\nsurfactant in the lipid disruption capability of the DNA surfactant\nconjugates generated from degradation of the NANs. Using NANs made\nwith different tail lengths (C<sub>12</sub> and C<sub>10</sub>), we\nshow that this mechanism likely holds true despite significant differences\nin the physical properties (i.e., critical micelle concentration (CMC),\nsurfactants per micelle, <i>N</i><sub>agg</sub>) of the\nresultant particles (C<sub>12</sub> and C<sub>10</sub> NANs). While\nthe total cellular uptake efficiencies of C<sub>12</sub> and C<sub>10</sub> NANs are similar, there were differences observed in their\ncellular distribution and localized trafficking, even after ensuring\nthat the total concentration of DNA was the same for both particles.\nUltimately, C<sub>10</sub> NANs appeared less diffuse within cells\nand colocalized less with lysosomes over time, achieving more significant\nknockdown of the target gene investigated, suggesting more effective\nendosomal escape. These differences indicate that the surfactant assembly\nand disassembly properties, including the number of surfactants per\nparticle and the CMC can have important implications for the cellular\ndelivery efficacy of DNA micelles and surfactant conjugates.