In some cases, image contrast and brightness was adjusted for clarity of presentation using ImageJ. three lead VLR candidates, VLR-Fc-11, VLR-Fc-30, and VLR-Fc-46 selectively target the brain Ononetin vasculature and traffic within brain microvascular FLT3 endothelial cells after intravenous administration in mice, with VLR-Fc-30 being confirmed as trafficking into the brain parenchyma. Epitope characterization indicates that the VLRs, in part, recognize sialylated glycostructures. These promising new targeting molecules have the potential for brain targeting and drug delivery with improved brain vascular specificity. test on n?=?3 replicates was used to determine statistical significance. (D) One-way ANOVA paired with Tukeys post-hoc analysis on n?=?3 replicates was used to determine statistical significance. *lectin, MAL II?=?lectin 2, ConA?=?Concanavalin A. To augment the glycan array results and demonstrate that glycan binding is involved in the observed interactions with MBECs, VLR-Fc binding to MBECs was quantified with and without pretreatment of the cells by 2-3,6,8 sialidase which cleaves terminal 2-3, 2-6, or 2-8 linked sialic acid residues from glycans (Fig.?6C). Binding of both VLR-Fc-11 and VLR-Fc-46 to MBECs was significantly decreased after treatment with sialidase. MBEC binding of VLR-Fc-30 upon sialidase treatment was also decreased but to a lesser extent. These results correlated well Ononetin with the glycoarray results where VLR-Fc -11 and -46 indicated a strong preference for a terminal sialic acid motif whereas the VLR-Fc-30 was less dependent on this motif. Moreover, the decrease in binding for VLR-Fc-11 and -30 upon sialidase treatment was less than that observed for Ononetin lectins with known sialic acid binding specificity, SNA and MAL II (Fig.?6C), suggesting additional glycan or proteinaceous components to the antigenic epitopes. To rule out VLR-Fc recognition of BBB receptors such as the transferrin receptor (rTfR), Insulin receptor (rIR), and low-density lipoprotein receptor (rLDLR) that have been extensively evaluated for their drug delivery capability, a competitive binding assay was employed. VLR-Fcs were pre-incubated with excess recombinant receptor ectodomains prior to a live MBEC cell surface binding assay. Ononetin MBEC binding was not affected for any of the VLR-Fcs upon receptor competition (Fig. S7). In contrast and as expected, competition with the rTfR protein reduced the anti-transferrin receptor Ononetin antibody (8D3) binding signal to?~?20% of the noncompetition signal, whereas rIR or rLDLR competition did not inhibit 8D3 binding. These results suggest that VLR-Fc-11, -30, and -46 do not bind TfR, IR, or LDLR. VLRs target and traffic the brain vasculature after systemic administration We next sought to determine whether VLR-Fc could target the BBB after systemic administration. To this end, VLR-Fc-11, -30, -46, and -192 were administered intravenously (IV) to mice at a dose of 10?mg/kg and allowed to circulate for 1?h. The mice were then perfused through the left heart ventricle with saline containing fluorescently labeled lectin to both clear the vasculature of unbound antibody and stain the luminal aspect of microvessels for subsequent imaging analysis. Examination of brain cryosections from VLR-Fc injected animals clearly indicated that all VLR-Fc clones except VLR-Fc-192 (Fig. S8) bound to the brain vasculature and co-localized with the perfused vascular lectin stain, as did the 8D3 anti-TfR IgG control (Fig.?7A). In contrast, the isotype control VLR-Fc-RBC36 exhibited negligible residual background, despite strong vascular lectin labeling, indicating perfusion was effective in removing any unbound circulating VLR-Fc. Therefore, vascular-localized VLR-Fc signal observed for VLR-Fc-11, -30, and -46 was a result of these VLR-Fc engaging their target antigens on the brain vasculature. To get an idea of brain selectivity of VLR-Fc targeting, uptake in the vascular beds of other organs was also.