Fig. 1: BT-CPA improves mRNA delivery of LNP after freeze-thaw (F-T).

a Schematic illustration showing the proposed mechanism of freezing-induced incorporation of betaine into LNPs. b The hydrodynamic diameters and polydispersity index (PDI) of LNPs (n = 3 technical replicates). Data are shown as mean ± standard deviation (SD). c A representative Cryogenic transmission electron microscopy (Cryo-TEM) image of LNP. The scale bar is 100 nm. The TEM imaging experiment was repeated independently three times. d The mRNA encapsulation of LNPs (n = 3 technical replicates). Data are shown as mean ± SD. e The mLuc expression in DC2.4 cells after treatment with free mRNA, LNP, or freeze-thawed LNPs with different cryoprotectants (CPAs) (n = 4 technical replicates). Data are shown as mean ± SD. Statistical significance was analyzed by one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. f A representative Cryo-TEM image of LNP+BT-CPA F-T. The scale bar is 100 nm. The TEM imaging experiment was repeated independently three times. g The mLuc expression in DC2.4 cells after treatment with free mRNA, LNP, or freeze-thawed LNP+BT-CPA with varying concentrations of betaine (n = 4 technical replicates). Data are shown as mean ± SD. h Representative IVIS images and i quantitative analysis of C57BL/6 mice following intramuscular injection of LNP or LNP+BT-CPA F-T at 4 and 24 h post-administration (n = 6 biologically independent samples). Data are shown as mean ± SD. Statistical significance was analyzed by unpaired two-tailed Student’s t-test. j Normalized mLuc expression in DC2.4 cells after treatment with LNPs formulated with ALC-0315, cKK-E12, and MC3 as ionizable lipids before and after F-T with BT-CPA (n = 4 technical replicates). Data are shown as mean ± SD. Statistical significance was analyzed by one-way ANOVA and Tukey’s multiple comparisons test. a was created in BioRender. Lu, X. (2025) https://BioRender.com/umtq3e5.