Lyophilization Strategies Enhancing the Storage Stability of RNA-loaded Nanoparticles

Lipid nanoparticles (LNPs) have emerged as versatile nanocarriers for RNA therapeutics, protecting them from nuclease-mediated degradation and allowing efficient cell uptake. One main challenge of LNPs is represented by their limited stability in aqueous suspensions, due to RNA release and/or LNP collapse over time. Lyophilization has been investigated as an effective method to produce dry RNA-loaded LNPs (RNA-LNPs) that can be stored at low temperatures for months. Particularly, cryo- and lyo-protectants have been used as additives to preserve RNA-LNP integrity, prevent aggregation phenomena and maintain RNA bioactivity during freeze-drying and storage. In our research, we designed novel polymer-lipid hybrid nanoparticles combining the superior stability of synthetic polymers in physiological media and the high RNA loading efficiency of lipids. Herein, we investigated lyophilization as an optimal method to preserve the physicochemical and biological properties of RNA-based nanomedicines. To this purpose we analyzed state-of-the art storage methods for RNA-LNPs and, then, studied optimal storage conditions for microRNA (miRNA)-loaded hybrid nanoparticles developed in our laboratory. Results from this work highlight the key role of lyophilization in the presence of cryo-/lyo-protectants as a means to enhance long-term storage of RNA-nanotherapeutics, which is expected to widen their accessibility for large-scale clinical use.