Fig. 4: Freezing induces content exchange between LNP and surrounding solutions.

a Schematic illustration of freeze-thawed LNP with GelRed. After freeze-thaw, GelRed enters LNP and binds with mRNA to emit fluorescence. b Fluorescence intensity of LNP, GelRed, LNP+GelRed, LNP+BT-CPA F-T+GelRed (GelRed was added after F-T), and LNP+BT-CPA+GelRed F-T (GelRed was added before F-T) (n = 3 technical replicates). Data are shown as mean ± SD. Statistical significance was analyzed by one-way ANOVA and Tukey’s multiple comparisons test. c Fluorescence intensity of LNP+BT-CPA+GelRed F-T, temperature decreasing rate with 1, 2.5, 5, 10, 15, 20 °C/min (n = 3 technical replicates). Data are shown as mean ± SD. d Schematic illustration of freeze-thawed fluorescein-loaded LNP. The encapsulated fluorescein in LNPs leaked out after F-T. e The concentration of encapsulated fluorescein in LNP before and after F-T. (n = 3 technical replicates). Data are shown as mean ± SD. Statistical significance was analyzed by unpaired two-tailed Student’s t-test. Figures 4a and 4d were created in BioRender. Lu, X. (2025) https://BioRender.com/d64j818.