Pulmonary delivery of siRNA lipoplexes and lipid nanoparticles using a vibrating mesh nebuliser

Inhalation via nebulisation is a promising method to deliver high concentrations of siRNA to the lung epithelium in a direct and non-invasive manner for the treatment of numerous respiratory-related illnesses. However, nebulisation can be destructive towards siRNA nanocarriers leading to loss of siRNA and a diminished therapeutic outcome. Herein, we sought to explore how the nebulisation process, including adjustments in aerosol droplet size impacts the physicochemical properties of several lipid-based siRNA nanocarrier formulations. These included PEGylated and non-PEGylated cationic DOTAP-based lipoplexes (LPXs) and C12-200 based lipid nanoparticles (LNPs). Two Aerogen® Pro vibrating mesh nebuliser devices with capacities to generate aerosols of differing volumetric mean diameters (VMD) were utilised. The aerosol droplet sizes for the different siRNA formulations were 4.80 to 4.89 µm (High VMD device) and 3.56 to 3.59 µm (Low VMD device) demonstrating that the emitted droplet size distribution was consistent across multiple siRNA nanocarrier types. Further, the formulations exhibited mass median aerodynamic diameters (MMAD) of 4.03 - 4.84 µm (High VMD device) indicating their potential for targeting siRNA lung deposition. Aggregation in both lipoplex formulations and a significant reduction in LNPs' siRNA encapsulation efficiency were observed. In vitro studies in Firefly luciferase (Fluc) expressing alveolar A549 cells demonstrated that cell viability and Fluc knockdown were generally unaffected by nebulisation. However, Fluc knockdown varied depending on formulation type and was highest for LNPs (93 %) and lowest for the PEGylated LPXs (max 30 %). Overall, this study shows that aerosols with consistent droplet size can be generated but the choice of nanocarrier impacts the stability and delivery efficacy and requires careful consideration for efficient nebulised siRNA delivery.