Fig. 3

In vitro characterization of multifunctional nanoparticles (MNPs). a Confocal microscopy images of 4T1 cells incubated with various formulations for 2 and 4 h. Scale bar, 100 nm. b, c Number of b DiD-positive and c FITC-positive 4T1 cells after incubation with DiD+FITC-OVA-N/A and DiD+FITC-OVA-S for 2 and 4 h (DiD, 1 μg/mL). Student’s t-test was performed. *P < 0.05. d Cell viability of 4T1 cells after incubation with different formulations for 24 h, as determined by the MTT assay. Blank nanoparticles were used as control. e Apoptosis of 4T1 cells stained with FITC-Annexin V and propidium iodide (PI) after incubation with different formulations for 24 h. f Percentage of cells in the apoptotic or necrotic stage after treatment with different formulations. Data are shown as mean ± SD (n = 3). Cells without any treatment were set as control group. CpG, immunopotentiator; CQ, chloroquine; DAPI, 4′,6-diamidino-2-phenylindole; DiD, 4-chlorobenzenesulfonate salt; FAM, fluorescein amidite; FITC, fluorescein; N, nanoparticles, N/A, nanoparticles coated with atezolizumab; OVA, ovalbumin; PTX, paclitaxel; S, solution