To test this hypothesis, we synthesized an Angiopep-2 construct functionalized at position R3 with biotin and at R1 and R2 with acetyl groups (3, Table , Supp. Fig. ). We then measured MoDE 3-dependent internalization of fluorescently labeled streptavidin in mouse brain endothelial cells (bEnd.3) and astrocytes (C8D1A) by flow cytometry. Astrocytes clear cellular debris in the CNS by phagocytosis and lysosomal degradation. Increasing cell-associated streptavidin(AF647) fluorescence was observed with increasing concentrations of MoDE 3 in both mouse brain cell lines (Figure ). No "hook" effect (inhibition of activity at higher concentrations of bifunctional molecule) was observed, which we hypothesize results from the high avidity of the tetravalent presentation of Angiopep-2 moiety when complexed to biotin, which would greatly slow its off rate. In both cell lines, endocytosis increased rapidly over 6 hours, and then began to plateau ( Figure ). also prepared with biotin at R2 (4) (Table , Supp. Fig. ) or R1 (5) (Table , Supp. Fig. ). Western blot analysis of the streptavidin monomer band showed that streptavidin concentration in the supernatant was reduced to 23  13% and 33  7% after treatment with MoDEs 3 and 4, respectively. No significant reduction in monomer or dimer bands was detected after treatment with MoDE 5, which was functionalized with biotin at R1. Importantly, MoDE-mediated endocytosis of streptavidin was dependent on the presence of biotin, as a peracetylated MoDE 6 (Table , Supp. Fig. ) did not induce endocytosis or depletion (Figure , Supp. Figure ) This activity was also dependent on cells, as no reduction in streptavidin signal was detected in a cell-free system (Supp. Figure ).