Fig. 4: In vivo safety and cell internalization profile of CP-LC-0729 LNP.

a Representative cryo-TEM images of CP-LC-0729 LNP. Scale bar, 100 nm. b Serum alanine aminotransferase levels (ALT), serum aspartate aminotransferase levels (AST), serum alkaline phosphatase levels (ALP), urea nitrogen concentration (UREA). Mice were intravenously injected (i.v) with mRNA-Luc-loaded LNPs at an mRNA dose of 2.5 mg/kg (n = 5 biologically independent samples). Serum was collected for ALT, AST, ALP and UREA analysis at 24 h and 48 h post-treatment. Data are presented as mean values ± SD. Two-way ANOVA with Tukey’s correction was used. c Body weight change for 15 days (measured made at days 0, 1, 2, 7, 9, 13 and 15). Mice were i.v injected with mRNA-Luc-loaded LNPs at an mRNA dose of 2.5 mg/kg (n = 5 biologically independent samples). Data are presented as mean values ± SD. d Uptake inhibition of CP-LC-0729 and MC3(DOPE) LNP by different endocytosis inhibitors (n = 3 biologically independent samples). The inhibitors used were Amiloride (inhibitor of macropinocytosis), Chlorpromazine (inhibitor of clathrin-mediated endocytosis), Genistein (inhibitor of caveolae mediated endocytosis), Methyl-β-cyclodextrin (inhibitor of lipid raft mediated endocytosis). Data are presented as mean values ± SD. e Hemolysis assay of CP-LC-0729 LNP and MC3(DOPE) LNP at pH 7.4 or 6.0. RBCs were incubated with CP-LC-0729 LNP and MC3(DOPE) LNP at an mRNA concentration of 2.5 μg/mL at 37 °C for 1 h. Positive and negative controls were carried out with 0.1% Triton-X and 1× PBS, respectively. Data are presented as mean ± SD (n = 3 biologically independent samples).