Fig. 4: In vitro BBB penetration, cellular and tumor uptake, and cytotoxicity of biomimetic nanogels.

a Cumulative transport ratio of ARNGs@ICG and RNGs@ICG across of the in vitro BBB trans-well model at different time (n = 3). Data are presented as mean ± SD (one-way ANOVA and Tukey’s multiple comparison test). b CLSM images of U87MG cells receiving various treatments and stained by fluorescein 5-isothiocyanate (FITC) and 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI). Scale bar: 20 μm. c Flow cytometry analysis of U87MG cells receiving various treatments. d Uptake of ARNGs@TMZ/ICG by U87MG multicellular spheroids after 4 h of incubation with and without NIR irradiation. Z-stack imaging was progressed from the bottom into the core of the spheroids at an interval of 10 µm. Scale bar: 500 μm; Top right: Schematic diagram of the 3D spherical U87MG model; Bottom right: Quantification of the relative ICG mean fluorescence intensity (MFI, n = 3). Data are presented as mean ± SD (one-way ANOVA and Tukey’s multiple comparison test). e CLSM images of DCFH-DA-stained U87MG cells after incubating with various nanogels for 4 h upon NIR irradiation. Scale bar: 100 μm. f Cell viability of U87MG cells by CellTiter-Lumi™ luminescent cell viability assay at 48 h after receiving various treatments (n = 5). Data are presented as mean ± SD (one-way ANOVA and Tukey’s multiple comparison test). g Apoptosis analysis of U87MG cells by flow cytometry at 48 h after receiving various treatments and stained by PI and Annexin V. For all studies, incubation time with treatment agents: 4 h; NIR: 808 nm, 0.5 W cm⁻², 5 min; ICG concentration: 10 µg mL⁻¹; TMZ concentration: 10 µg mL⁻¹.