Angiopep-2 is often incorporated in nanoparticles to aid in transcytosis of therapeutics across the BBB. To mimic the BBB, we used bEnd.3 cells cultured as a monolayer on a porous membrane (Figure , Supp. Figure ). The bEnd.3 cell monolayer reduced spontaneous migration of 70kDa FITC-Dextran between the two chambers by 95.7  1.0% compared to the no cell control (Supp. Figure ). Transcytosis was assessed both in apical-to-basal and basal-to-apical directions (Figure ). Over 24h, we observed MoDE 3-mediated uptake of streptavidin into the cell monolayer, as well as transport into the basal (Figure ) and apical (Figure ,F) chamber. The apparent permeability (Supp. was significantly higher in the presence of MoDE 3 than its absence (Figure ), and the effective permeability of MoDE 3 transport was not significantly different from the positive control for receptor-mediated transcytosis, transferrin, suggesting an active mechanism of transcytosis. Conversely, streptavidin transport in the absence of MoDE occurred at a similar rate to FITC-dextran, likely due to passive diffusion across the cell monolayer (Supp. Table , Supp. Figure ). This corresponds with the nonspecific background migration of protein. Combined, this work demonstrates that MoDEs can both degrade proteins via the lysosomal pathway and enable transcytosis across brain endothelial cells.