ABSTRACT
Although mRNA lipid nanoparticles (LNPs) are highly effective as vaccines, their efficacy for pulmonary delivery has not yet fully been established. A major barrier to this therapeutic goal is their instability during aerosolization for local delivery. This imparts a shear force that degrades the mRNA cargo and therefore reduces cell transfection. In addition to remaining stable upon aerosolization, mRNA LNPs must also possess the aerodynamic properties to achieve deposition in clinically relevant areas of the lungs. We addressed these challenges by formulating mRNA LNPs with SM-102, the clinically approved ionizable lipid in the Spikevax COVID-19 vaccine. Our lead candidate, B-1, had the highest mRNA expression in both a physiologically relevant air-liquid interface (ALI) human lung cell model and in healthy mice lungs upon aerosolization. Further, B-1 showed selective transfection in vivo of lung epithelial cells compared to immune cells and endothelial cells. These results show that the formulation can target therapeutically relevant cells in pulmonary diseases such as cystic fibrosis. Morphological studies of B-1 revealed differences in the surface structure compared to LNPs with lower transfection efficiency. Importantly, the formulation maintained critical aerodynamic properties in simulated human airways upon next generation impaction. Finally, structure-function analysis of SM-102 revealed that small changes in the number of carbons can improve upon mRNA delivery in ALI human lung cells. Overall, our study expands the application of SM-102 and its analogs to aerosolized pulmonary delivery and identifies a potent lead candidate for future therapeutically active mRNA therapies.
ABSTRACT
Fibrin fibers form the structural backbone of blood clots; fibrinolysis is the process in which plasmin digests fibrin fibers, effectively regulating the size and duration of a clot. To understand blood clot dissolution, the influence of clot structure and fiber properties must be separated from the effects of enzyme kinetics and perfusion rates into clots. Using an inverted optical microscope and fluorescently-labeled fibers suspended between micropatterned ridges, we have directly measured the lysis of individual fibrin fibers. We found that during lysis 64 ± 6% of fibers were transected at one point, but 29 ± 3% of fibers increase in length rather than dissolving or being transected. Thrombin and plasmin dose-response experiments showed that the elongation behavior was independent of plasmin concentration, but was instead dependent on the concentration of thrombin used during fiber polymerization, which correlated inversely with fiber diameter. Thinner fibers were more likely to lyse, while fibers greater than 200 ± 30 nm in diameter were more likely to elongate. Because lysis rates were greatly reduced in elongated fibers, we hypothesize that plasmin activity depends on fiber strain. Using polymer physics- and continuum mechanics-based mathematical models, we show that fibers polymerize in a strained state and that thicker fibers lose their prestrain more rapidly than thinner fibers during lysis, which may explain why thick fibers elongate and thin fibers lyse. These results highlight how subtle differences in the diameter and prestrain of fibers could lead to dramatically different lytic susceptibilities.
