Freeze-Drying in Sucrose Followed by Cryomilling Enables the Formulation of sa-mRNA-LNP Powders for Inhalation

Background: Self-amplifying mRNA (sa-mRNA) represents a promising platform for vaccines and gene therapies, offering sustained protein expression at low doses through self-replication. For vaccines targeting respiratory pathogens, pulmonary delivery of sa-mRNA lipid nanoparticles (LNPs) is particularly advantageous, enabling direct delivery to the Infection site and induction of mucosal immunity. Objective: In this study, we evaluated the stability of sa-mRNA-LNPs under refrigerated and frozen conditions and developed a dry powder formulation suitable for inhalation, produced by freeze-drying followed by cryomilling with leucine. Methods: sa-mRNA-LNPs formulated in HEPES buffer with 20% (w/v) sucrose were stored for up to 8 weeks as liquid or freeze-dried samples at various temperatures (-80 °C, -20 °C, 4 °C, and 20 °C). Biological stability was assessed by transfection efficiency in HeLa cells, while physical stability was characterized by encapsulation efficiency, zeta potential, particle size, and polydispersity index. Results: Liquid formulations remained stable for at least 8 weeks at -80 °C and -20 °C but rapidly lost stability at 4 °C and 20 °C. Freeze-drying effectively preserved sa-mRNA-LNP functionality and structural integrity for up to 8 weeks at 4 °C, with only minor structural changes. Subsequent cryomilling in the presence of 4 wt-% leucine produced a respirable dry powder while retaining approximately 60% of the original sa-mRNA-LNP functionality. Although cryomilling induced some structural alterations, the remaining functional fraction remained stable during storage. The resulting powders displayed favorable aerosol performance for deep lung delivery, as demonstrated by cascade impaction (MMAD = 4.13 ± 0.26 µm). Conclusions: In conclusion, freeze-drying effectively preserved sa-mRNA-LNP integrity at 4 °C, whereas cryomilling with leucine produced a respirable dry powder suitable for pulmonary delivery, providing a foundation for globally accessible, needle-free sa-mRNA vaccines against respiratory diseases.

Cyclops DPI; cryomilling; freeze-drying; freezing; inhalation; lyophilization; sa-mRNA–LNPs; stabilization; sucrose.