Figure 4: Analysis of angiopep-2 transport in BBB spheroid.

(a) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. (b) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. (c) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. (d) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni’s multiple comparison test. (e) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. (f) Bar graph quantifying the transport of angiopep-2 (10 μM; from e). Statistical analyses were performed using the Student’s t-test. (g) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. (h) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g). Statistical analyses were performed using the one-way ANOVA and Tukey’s multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars (n_spheroid=10, n_experiment=3). (i) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml⁻¹) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars (n_spheroid=3–6, n_experiment=3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett’s multiple comparison test. (j) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.