In vitro BBB transcytosis and in vitro escape from abluminal LRP1-mediated clearance. (A) Uptake of various NPs in normal BBB endothelial cells. Data are presented as mean ± SD (n = 3). (B) Uptake in bend.3 cells, NPs-K-s-A were pre-incubated in PBS/MCM mixture with different ratios for 2 h. Data are presented as mean ± SD (n = 4). (C) NPs-K-s-A was pre-incubated in PBS/MCM (1:9, v/v) for different time with different concentration MMP inhibitor BB-94. Data are presented as mean ± SD (n = 4). (D) NPs-K-s-A and NPs-K-i-A were pre-incubated in PBS/MCM (1:9, v/v) for different time with or without 5 μmol/L BB-94. Data are presented as mean ± SD (n = 4). (E) Qualitative uptake of DOX-loaded NPs with FITC-labeled K-s-A in normal medium or MCM in bEND.3 cells imaged using confocal microscope. Scale bar = 100 μm. (F) Left: Schematic illustration of the in vitro BBB model for studying backward BBB passing ability. Right: Distribution of IR780-loaded NPs-K-s-A and NPs-K-i-A in blood side, brain endothelium, and brain side. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.