Fig. 4

Intracellular toxicity evaluation and amplification effects of cGAS-STING signaling pathway. (A) The cellular uptake of MPCZ NPs at different concentrations was analyzed by ICP-MS. The labeled asterisk represents statistical significance compared with control group. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) – (D) Cell viability analysis of 4T1 cells treated by PBS, MPC, MPCZ NPs (comparable to 25, 50, 100 and 200 µg/mL MPC or 1.25, 2.5, 5.0 and 10.0 µg/mL ZPP) (n = 3). MTT (B), Live/dead staining (C) and Fluorescein-annexin V and PI staining assays (D) of 4T1 cells treated by PBS, MPC, MPCZ NPs (comparable to 100 µg/mL MPC or 5.0 µg/mL ZPP) for 6 h, then irradiated with or without 808 nm laser (1 W/cm²) for 10 min and incubated for following 18 h. Labeled asterisk represents statistical significance compared with MPC NPs without 808 nm laser irradiation via one-way ANOVA with the Tukey post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. (E) The protein expression levels of STING, p-STING, p-TBK1 and TBK1 were analyzed by western blot in 4T1 cells. (F) The release of INF-β from PBS, MPC NPs and MPCZ NPs with or without 808 nm laser irradiation detected by ELISA kit (n = 3). For (E) and (F), cells were treated by PBS, MPC NPs and MPCZ NPs (comparable to 25 µg/mL MPC or 1.25 µg/mL ZPP) for 6 h and irradiated with or without 808 nm laser (1 W/cm²) for 10 min for following 6 h incubation. Based on one-way ANOVA with Tukey post-hoc analysis, the asterisks represent statistical significance in comparison to the PBS group. *p < 0.05, ***p < 0.001. (G) Schematic illustration of the 4T1/DCs transwell system treated with different NPs. (H) Flow cytometry measurements of DCs surface for CD86 and CD80 expression after different treatments