Figure 3.

Effects of statins on the expression of LDL receptors and on the permeability of LDL receptor-targeted liposomal doxorubicin across the BBB. The hCMEC/D3 cells were incubated in the absence (CTRL) or presence of 0.1 µmol·L⁻¹mevastatin (MVS) or simvastatin (SIM) for 24 h (A, B), 48 h (B, D and E), then subjected to the following investigations. (A) RT-PCR of LDL receptors. Total RNA was extracted, reverse transcribed and amplified by RT-PCR, as indicated in Methods. Measurements were performed in triplicate and data are presented as means ± SD (n= 3). Versus CTRL: *P < 0.005. (B) Flow cytometry analysis of surface LDL receptors in untreated cells (grey peak) or treated with MVS (continuous line) or SIM (dotted line) for 48 h. The figures shown here are representative of 3 similar experiments, performed in triplicate. (C) Apo-Lipodox imaging by TEM. The micrographs (28 500× magnification) are representative of three similar experiments. (D) Transport of apo-Lipodox across the BBB monolayer. The hCMEC/D3 cells were grown to the confluence in Transwell insert, in fresh medium or in the presence of statins or of the NO scavenger PTIO (100 µmol·L⁻¹ for 48 h), then 5 µmol·L⁻¹ apo-Lipodox was added in the upper chamber. After 3 h, the amount of the drug recovered by the lower chamber medium was measured fluorimetrically. Measurements were performed in duplicate and data are presented as means ± SD (n= 4). Versus CTRL: *P < 0.005; versus condition without PTIO: °P < 0.001. (E) Transport of Lipodox and apo-Lipodox across the BBB monolayer. The hCMEC/D3 cells were grown up to the confluence in Transwell insert, in fresh medium or in the presence of SIM, then 5 µmol·L⁻¹ Lipodox or apo-Lipodox was added in the upper chamber. When indicated, the free LDL receptor-targeted apoB100 peptide (the same that was conjugated with apo-Lipodox), was co-incubated with the liposomes (100 µmol·L⁻¹; B100). After 3 h, the amount of doxorubicin recovered by the lower chamber medium was measured fluorimetrically. Measurements were performed in duplicate and data are presented as means ± SD (n= 3). Versus CTRL: *P < 0.005; versus SIM: °P < 0.05.