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. 2021 Sep 6;10(20):2100812. doi: 10.1002/adhm.202100812

Figure 4.

Figure 4

Evaluation of diphtheria toxin mediated transcytosis via BBB. A) TEM images of the trans‐well membrane with hCMEC/D3 cells after 4 h incubation with CRM‐197‐nanogel‐AF488 in co‐culture with U‐87 cells (left, 4600‐fold magnification) and TEM images of the trans‐well membrane with pericytes after 4 h incubation with CRM‐197‐nanogel‐AF488 in co‐culture with U‐87 cells (right, 6000‐fold magnification). B) Fluorescent microscope images of nanogel‐AF488 with CRM‐197 (CRM‐197‐NG‐AF488) and without CRM‐197 (NG‐AF488) in basolateral seeded NHA or U‐87 cells after 4 h incubation with the BBB model (blue: DAPI staining, 66‐fold magnification). C) Gamma‐counter analysis for nanogel transcytosis and uptake quantification. Radiolabeled functionalized and non‐functionalized nanogel accumulation was measured in hCMEC/D3 cells, basolateral media, and seeded NHA after 90 min incubation with 1°MBq per well (n = 5). D) Gamma‐counter analysis for nanogel transcytosis and uptake quantification. Radiolabeled functionalized and non‐functionalized nanogel accumulation was measured in hCMEC/D3 cells, basolateral media, and seeded U‐87 cells after 90 min incubation. For inhibition study, the samples were pre‐treated with the CRM‐197 protein (0.1°mg per well for 60 min) (****p < 0.001; ***p < 0.005, students t‐test).