Optimization of ionizable lipids for aerosolizable mRNA lipid nanoparticles.

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.

Controlled loading of albumin-drug conjugates ex vivo for enhanced drug delivery and antitumor efficacy.

To harness the intrinsic transport properties of albumin yet improve the therapeutic index of current in situ albumin-binding prodrugs, we developed albumin-drug conjugates with a controlled loading that achieved better antitumor efficacy. Here, model drug monomethyl auristatin E (MMAE) was conjugated ex vivo to Cys34 of albumin via a cathepsin B-sensitive dipeptide linker to ensure that all drug would be bound specifically to albumin. The resulting albumin-drug conjugate with a drug to albumin ratio (DAR) of 1 (ALDC1) retained the native secondary structure of albumin compared to conjugate with a higher DAR of 3 (ALDC3). ALDC1 exhibited improved drug release and cytotoxicity compared to ALDC3 in vitro. Slower plasma clearance and increased drug exposure over time of ALDC1 were observed compared to ALDC3 and MMAE prodrug. In single dose studies with MIA PaCa2 xenografts, cohorts treated with ALDC1 had the highest amount of MMAE drug in tumor tissues compared to other treatment arms. After multiple dosing, ALDC1 significantly delayed the tumor growth compared to control treatment arms MMAE, MMAE-linker conjugate and ALDC3. When dosed with the maximum tolerated dose of ALDC1, there was complete eradication of 83.33% of the tumors in the treatment group. Ex vivo conjugated ALDC1 also significantly inhibited tumor growth in an immunocompetent syngeneic mouse model that better recapitulates the phenotype and clinical features of human pancreatic cancers. In summary, site-specific loading of drug to albumin at 1:1 ratio allowed the conjugate to better maintain the native structure of albumin and its intrinsic properties. By conjugating the drug to albumin prior to administration minimized premature cleavage and instability of the drug in plasma and enabled higher drug accumulation in tumors compared to in situ albumin-binding prodrugs. This strategy to control drug loading ex vivo ensures complete drug binding to the albumin carrier and achieves excellent antitumor efficacy, and it has the potential to greatly improve the outcomes of anticancer therapy.

Physical determinants of fibrinolysis in single fibrin fibers.

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.