Fig. 4: Cellular uptake, cytotoxicity, and tumor spheroid penetration of DMSN@Pla-Lipo.

A CLSM images of COLO205 cells after 3 h incubation with MSN, MSN@Lipo, or MSN@Pla-Lipo. The cell membrane was stained with DiO (green), and MSN was labeled with iFluor 647 (red). Bar, 10 μm. B Quantified cell-associated iFluor 647 fluorescence intensity after cellular uptake in panel A using flow cytometry (n = 3 independent experiments). C Cell viabilities after treatment with DMSN, DMSN@Lipo, or DMSN@Pla-Lipo for 24 h were examined using CCK-8 assay (n = 5 independent experiments). D The cells after the indicated treatments (DOX, 1 μg/mL) for 24 h were stained with calcein-AM and propidium iodide (PI) for simultaneous fluorescence imaging of viable and dead cells. Bar, 50 μm. E Penetration of iFluor 647-labled MSN, MSN@Lipo, or MSN@Pla-Lipo in COLO205 tumor spheroids (iFluor 647, 800 ng/mL). The area marked with blue circles were considered the inside area. Bar, 100 μm. F Quantified fluorescence intensity at the 250 μm cross-section in panel E (n = 3 independent experiments). G Cytotoxicity of the various formulations in COLO205 tumor spheroids, which were stained with calcein-AM and PI. Representative micrographs were shown, and the tests were repeated three times. Bar, 100 μm. H Flow cytometry assay of apoptotic tumor cells in panel G. I Statistical assay of cell apoptosis rate in panel H. Cell apoptosis rate refers to the ratio of annexin V-positive cells to the total analyzed cells (n = 3 independent experiments). Data in B, C, F, I are presented as mean ± s.d. P-values were calculated by one-way ANOVA with Tukey’s multiple comparisons test B, C, F, I.