FIGURE 2.

Design, synthesis, and characterization of a BBB organoid–penetrating supramolecular cationic inhibitor of Hsp90. (A) The schematic shows the design of supramolecular drug vehicles using lipids with varying headgroup charge: AN01 and AN02. (B) The schematic depicts the BBB organoid model used for in vitro experiments. The endothelial cell layer (hCMEC) surrounds the pericyte and astrocyte layers of cells. (C) The immunofluorescent image shows infiltration of AN01 and AN02 into the BBB organoid. The green signal indicates the amount of lipid penetrating the endothelial barrier deep enough to account for passage into the BBB organoid (80 μm). Scale bar = 50 μm. The right panel quantifies the fluorescent intensity at a depth of 80 μm. (D) The schematic shows the design of SCI-101, comprising cholesterol-radicicol conjugates assembled together with positively charged lipids into a supramolecular structure. (E) The representative histogram shows the size distribution of SCI-101 (upper panel) and longitudinal trace of average size over 120 days to demonstrate stability of the drug vehicles (lower panel). (F) The schematic describes the experimental design for qualification of radicicol in the BBB organoid. The right panel bar graph quantifies the penetration of radicicol free drug or SCI-101 infiltrated into the BBB organoid as measured by the area under the curve. (G) Experimental design schematic for drug washout studies. Following 24 h exposure to either free drug radicicol or SCI-101 at equivalent concentration (5 μm), the cells were washed and recovered with fresh media for 72 h. The right panel histogram quantifies the proliferation of U-87 cells 72 h after drug washout. The values shown are relative to the number of cells prior to drug washout in the vehicle control group. ***p<0.001 by ordinary one-way ANOVA with Tukey’s multiple-comparison test.