Freezing induced incorporation of betaine in lipid nanoparticles enhances mRNA delivery
Xingdi Cheng, Xia Zheng, Kun Tao, Haonan Huo, Zhang Liu, Xueguang Lu, Jianjun Wang
Abstract
Lipid nanoparticles (LNPs) are key non-viral carriers for mRNA vaccines and therapeutics, but the inherent instability of mRNA necessitates sub-zero storage with cryoprotectants (CPAs) to prevent freeze-induced LNP aggregation and compromised mRNA delivery. Here we show that ice formation during freezing concentrates CPAs with LNPs in the remaining liquid—a phenomenon known as freeze concentration. This creates a steep concentration gradient of CPAs across the lipid membrane that drives passive CPAs diffusion into LNPs. By leveraging this process, we developed betaine-based CPAs that both preserve the stability of LNP and enter LNP during freeze-thaw. The incorporated betaine enhances endosomal escape and boosts mRNA delivery of LNP. In female mice, betaine-loaded LNPs elicit stronger humoral and cellular immune responses, providing dose-sparing advantages. These findings highlight freeze concentration as a promising LNP formulation strategy and underscore the role of CPA as active modulators of LNP structure and function. Cryoprotectants are needed for the storage of lipid nanoparticles (LNPs). Here, the authors report on a study which shows that freezing can be harnessed to reformulate LNPs in a way that both preserves their integrity and boosts mRNA delivery after sub-zero storage by increasing endosomal escape.