During the pandemic, lipid nanoparticles (LNPs) became widely established as RNA nanocarriers, and hold the promise of future targeting of a broad variety of previously untreatable diseases. LNPs are mostly administered invasively via intramuscular or intravenous injections. Given the lung's large surface, high vascularization and low nuclease abundance, inhalation offers a promising alternative for both local and systemic delivery of LNPs. Vibrating mesh nebulizers present a patient-friendly, high-dose delivery platform. However, the nebulization process imposes thermal and mechanical stress on the LNP formulation. This study contributes to a better understanding of how nebulization affects the physicochemical properties and biological activity of LNPs, depending on formulation and process parameters. We investigated the impact of formulation and process variables such as temperature, concentration, buffer type, and RNA modality on LNP properties including particle size distribution, zeta potential, in vitro activity, and RNA integrity. While aggregating, siRNA LNPs protected the encapsulated RNA from degradation, and preserved biological function. In contrast, after the nebulization of mRNA LNPs the cargo was degraded and the biological function diminished. This observation can possibly be attributed both to the higher sensitivity of mRNA toward physical and chemical degradation, and the cargo-dependent morphology of LNPs. While demonstrating that siRNA LNPs preserved their most important characteristics, namely RNA integrity and biological function, our findings emphasize the need for route-specific optimization of LNPs, which need to meet different critical quality criteria when used for inhalation rather than injection.
