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18 reviewsLipid nanoparticles (LNPs) are key non-viral carriers for mRNA vaccines andtherapeutics, but the inherent instability of mRNA necessitates sub-zero storage with cryoprotectants (CPAs) to prevent freeze-induced LNP aggregation1234567890():,;1234567890():,;and compromised mRNA delivery. Here we show that ice formation duringfreezing concentrates CPAs with LNPs in the remaining liquid—a phenomenonknown as freeze concentration. This creates a steep concentration gradient ofCPAs 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 incorporatedbetaine enhances endosomal escape and boosts mRNA delivery of LNP. Infemale mice, betaine-loaded LNPs elicit stronger humoral and cellular immuneresponses, providing dose-sparing advantages. These findings highlight freezeconcentration as a promising LNP formulation strategy and underscore therole of CPA as active modulators of LNP structure and function.