The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability, and Safety of Lipid‐Based Nanoparticles for mRNA and DNA Delivery

Lipid-based nanoparticles have recently shown great promise, establishing themselves as the gold standard in delivering novel RNA therapeutics. However, research on the effects of storage on their efficacy, safety, and stability is still lacking. Herein, the impact of storage temperature on two types of lipid-based nanocarriers, lipid nanoparticles (LNPs) and receptor-targeted nanoparticles (RTNs), loaded with either DNA or messenger RNA (mRNA), is explored and the effects of different cryoprotectants on the stability and efficacy of the formulations are investigated. The medium-term stability of the nanoparticles was evaluated by monitoring their physicochemical characteristics, entrapment and transfection efficiency, every two weeks over one month. It is demonstrated, that the use of cryoprotectants protects nanoparticles against loss of function and degradation in all storage conditions. Moreover, it is shown that the addition of sucrose enables all nanoparticles to remain stable and maintain their efficacy for up to a month when stored at -80 °C, regardless of cargo or type of nanoparticle. DNA-loaded nanoparticles also remain stable in a wider variety of storage conditions than mRNA-loaded ones. Importantly, these novel LNPs show increased GFP expression that can signify their future use in gene therapies, beyond the established role of LNPs in RNA therapeutics.