Fig. 5

In vitro characterization of ApoLNP (a) ApoLNP was prepared by encapsulating the SMAC-P-FRRG-DOX prodrug inside the 10PS. b The formed ApoLNP (1 mg/mL) was assessed via DLS and cryoTEM, confirming a similar size and morphology to that before SMAC-P-FRRG-DOX loading. c The dispersion stability of ApoLNP in PBS buffer (1 mg/mL) was observed for up to 6 days using DLS. d The ApoLNP (15 mg/mL) was placed into a dialysis bag (MWCO = 12–14 kDa) and dialyzed with 10 mL PBS for 10 days to examine the release behavior of SMAC-P-FRRG-DOX. e The HPLC analysis of SMAC-P-FRRG-DOX (0.1 mM) before and after the incubation with cathepsin B (10 μg/mL) displayed its cathepsin B-specific cleavage into SMAC-P-FRR and G-DOX (f) The excessive cathepsin B expression level of 4T1 cancer cells compared to that of H9C2 normal cells was analyzed via western blot assay (n = 3). g 4T1 cells were treated with ApoLNP (5 μM, based on SMAC-P-FRRG-DOX content) and their CLSM analysis was carried out at 1, 6, and 24 h after the treatment to determine its endocytosis and intracellular distribution. h The ratios of LNPs (Flamma 648-PE, red color) and DOXs (green color) localized inside the cytosol and nuclei were compared to confirm the increased intranuclear distribution of DOXs over time (n = 3). i 4T1 cancer and H9C2 normal cells were exposed to various concentrations of ApoLNPs, DOXs, and 10PSs from 0.01 to 200 μM and their CCK-8 assay was conducted to prove the selective toxicity of ApoLNP to cathepsin B-overexpressing cancer cells. j The reduced IAP expression level of ApoLNP-treated 4T1 cells over DOX-treated ones was determined via the western blot (n = 3). * p < 0.05, **** p < 0.0001