Fig. 2: Construction of NIF LNPs with improved mRNA transfection efficiency.

a Adding NAIPs in PL32 LNPs to prepare non-inflammatory NAIP LNPs. b In vitro expression of mLuc delivered by NAIP-LNPs formulations in A549 cells. 24 hours after incubation with mLuc LNPs, the expression was measured using a BioTek plate reader (n = 3 biologically independent wells). c Chemical structures of the lead candidate NAIPs. d Expression of mLuc 6 h after intratracheal administration of the representative NAIP-LNPs and the original PL32 LNPs (0.2 mg/kg, n = 4 mice). Lung tissues were lysed, and luminescence was detected using a BioTek plate-reader. The quantification of the mLuc expression was presented on the right in (e), P-CAL: PL32 with calycosin; P-OA: PL32 with oleanolic acid; P-AA: PL32 with asiatic acid; P-UA: PL32 with ursolic acid, (n = 4 mice). f Relative mRNA expression of Il-1β and Cxcl1 in lung tissues of (d) (n = 3 mice). g Quantification of neutrophils, macrophages, and M1-/M2-macrophages by flow cytometry analysis in previous lung tissues of (d) (n = 3 mice). h, i Inflammatory cytokines levels of BAL in idiopathic pulmonary fibrosis (IPF) and acute lung injury (ALI) mice intratracheally instilled with PL32 LNPs and P-UA LNPs for 24 h (0.6 mg/kg, n = 3 mice per group). j Anatomical inspection and H&E stain of lung tissues. Scale bar, 100 μm. k Detection of inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) treated with PL32 LNPs and P-UA LNPs for 24 h by Cytometric Bead Array (CBA) - I Kit (n = 3 independent wells). l Schematic illustration of mRNA expression and inflammation in lung after administration of non-inflammatory P-UA LNPs and traditional PL32 LNPs. Data were represented as mean ± SEM. Statistical significance was calculated through two-tailed unpaired Student’s t-test. (h, i) One-way ANOVA with Dunnett test (e–g), and One-way ANOVA with Tukey’s test (b, k). Figure 2a, k, l were created in BioRender. com with attribution line Miao, L. (2025) https://BioRender.com/4tk9uu8, Miao, L. (2025) https://BioRender.com/7t6a86q Miao, L. (2025) and Miao, L. (2025) https://BioRender.com/9bx34ve), respectively. Source data are provided as a Source Data file.