In Vitro (Transwell) BBB Permeability of Vectors

Concentration of nanoconjugates in the apical compartment was 4.55 μM, corresponding to 0.7 μM in the blood of mice injected in dose 0.125X (see definition in Table S2). Apical concentrations of the inhibitors were 10 μM of JPH203, 10 μM of AP2, and 0.219 ng/mL of Aβ peptide (mimicking the peptide levels in healthy blood).³⁹ The basolateral concentration of Aβ peptide was 9.8 ng/mL.³⁹ Samples were taken at multiple time points until 3 h from the addition of nanoconjugates, and fluorescence was measured to calculate endothelial permeability. The permeation of P/LLL/D3, P/LLL/AP2, and P/LLL(40%) through the human brain microvascular endothelial cell monolayer was tested in the presence of the inhibitors. Analysis confirmed that TJ proteins remained intact, as indicated by the maintenance of trans-endothelial electric resistance (TEER) (Supporting Information Figure S3). The MTS assay at nanoconjugate concentrations of 9.1, 18.2, and 36.4 μM (6.7-fold above the blood levels after in vivo injection of doses 0.25X, 0.5X, and 1X in Table S2) showed that none of the nanoconjugates significantly decreased cell viability in the presence of the in vivo equivalent of 1X dosage.