One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc–siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc–siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.

We present a theoretical model for predicting equilibrium wetting configurations of two-dimensional droplets on periodically grooved hydrophobic surfaces. The main advantage of our model is that it accounts for pinning/depinning of the contact line at step edges, a feature that is not captured by the Cassie and Wenzel models. We also account for the effects of gravity (via the Bond number) on various wetting configurations that can occur. Using free-energy minimization, we construct phase diagrams depicting the dependence of the wetting modes (including the number of surface grooves involved in the wetting configuration) and their corresponding contact angles on the geometrical parameters characterizing the patterned surface. In the limit of vanishing Bond number, the predicted wetting modes and contact angles become independent of drop size if the geometrical parameters are scaled with drop radius. Contact angles predicted by our continuum-level theoretical model are in good agreement with corresponding results from nanometer-scale molecular dynamics simulations. Our theoretical predictions are also in good agreement with experimentally measured contact angles of small drops, for which gravitational effects on interface deformation are negligible. We show that contact-line pinning is important for superhydrophobicity and that the contact angle is maximized when the droplet size is comparable to the length scale of the surface pattern.

We used molecular dynamics (MD) simulations to study the wetting of Lennard-Jones cylindrical droplets on surfaces patterned with grooves. By scaling the surface topography parameters with the droplet size, we find that the preferred wetting modes and contact angles become independent of the droplet size. This result is in agreement with a mathematical model for the droplet free energy at small Bond numbers for which the effects of gravity are negligible. The MD contact angles for various wetting modes are in good agreement with those predicted by the mathematical model. We construct phase diagrams of the dependence of the wetting modes observed in the MD simulations on the topography of the surface. Depending on the topographical parameters characterizing the surface, multiple wetting modes can be observed, as is also seen experimentally. Thus, our studies indicate that MD simulations can yield insight into the large-length-scale behavior of droplets on patterned surfaces.

Superhydrophobic surfaces often incorporate roughness on both micron and nanometer length scales, although a satisfactory understanding of the role of this hierarchical roughness in causing superhydrophobicity remains elusive. We present a two-dimensional thermodynamic model to describe wetting on hierarchically grooved surfaces by droplets for which the influence of gravity is negligible. By creating wetting phase diagrams for droplets on surfaces with both single-scale and hierarchical roughness, we find that hierarchical roughness leads to greatly expanded superhydrophobic domains in phase space over those for a single scale of roughness. Our results indicate that an important role of the nanoscale roughness is to increase the effective Young's angle of the microscale features, leading to smaller required aspect ratios (height to width) for the surface structures. We then show how this idea may be used to design a hierarchically rough surface with optimally high contact angles.

The wetting configuration of a liquid droplet on a rough or physically patterned surface is typically characterized by either the Cassie wetting mode, in which the droplet resides on top of the roughness, or the Wenzel mode, in which the droplet penetrates into the roughness. For a fixed surface topology and droplet size, one of these modes corresponds to the global free-energy minimum. However, the other state is often metastable and long-lived due to a free-energy barrier that hinders the transition between the two wetting states. Metastable wetting states have been observed experimentally, and we also observe them in molecular dynamics (MD) simulations of a droplet on a grooved surface. Using forward flux sampling, we study the kinetics of the Cassie to Wenzel and Wenzel to Cassie transitions for two-dimensional droplets on periodically grooved substrates. The global-minimum wetting states that emerge from our nanoscale MD approach are consistent with those predicted by a macroscopic model based on free energy minimization. We find that the free-energy barriers for these transitions depend on the droplet size and surface topology. A committor analysis indicates that the transition-state ensemble consists of droplets that are on the verge of initiating/breaking contact with the substrate at the bottom of the grooves.