ApoE-Targeting Increases the Transfer of Solid Lipid Nanoparticles with Donepezil Cargo across a Culture Model of the Blood-Brain Barrier

Abstract

Pharmacological treatment of central nervous system (CNS) disorders is difficult, because the blood-brain barrier (BBB) restricts the penetration of many drugs into the brain. To solve this unmet therapeutic need, nanosized drug carriers are the focus of research efforts to develop drug delivery systems for the CNS. For the successful delivery of nanoparticles (NPs) to the brain, targeting ligands on their surface is necessary. Our research aim was to design a nanoscale drug delivery system for a more efficient transfer of donepezil, an anticholinergic drug in the therapy of Alzheimer's disease across the BBB. Rhodamine B-labeled solid lipid nanoparticles with donepezil cargo were prepared and targeted with apolipoprotein E (ApoE), a ligand of BBB receptors. Nanoparticles were characterized by measurement of size, polydispersity index, zeta potential, thermal analysis, Fourier-transform infrared spectroscopy, in vitro release, and stability. Cytotoxicity of nanoparticles were investigated by metabolic assay and impedance-based cell analysis. ApoE-targeting increased the uptake of lipid nanoparticles in cultured brain endothelial cells and neurons. Furthermore, the permeability of ApoE-targeted nanoparticles across a co-culture model of the BBB was also elevated. Our data indicate that ApoE, which binds BBB receptors, can potentially be exploited for successful CNS targeting of solid lipid nanoparticles.

Keywords: ApoE; blood–brain barrier; donepezil; drug delivery to brain; solid lipid nanoparticle; targeted drug delivery.

Figure 1

(a) Schematic drawing of non-targeted (DON-SLN) and ApoE targeted (APOE-DON-SLN) donepezil (DON) encapsulated solid lipid nanoparticles (SLN). (b) Main physico-chemical properties of SLNs. Values presented are means ± SD. EE%: encapsulation efficiency. (c) Transmission electron microscopy images of DON-SLNs and APOE-DON-SLNs. Scale bars: 100 nm for both DON-SLN and APOE-DON-SLN groups.

Figure 2

In Vitro donepezil HCl release profiles from donepezil aqueous solution (DON), non-targeted SLNs (DON-SLN), and SLNs functionalized with ApoE (APOE-DON-SLN).

Figure 3

DSC thermograms of dynasan 116, donepezil HCl (DON), non-targeted (DON-SLN), and targeted SLNs (APOE-DON-SLN).

Figure 4

FTIR spectra of (a) dynasan 116, (b) donepezil HCl (DON), (c) non-targeted (DON-SLN), and (d) targeted SLNs (APOE-DON-SLN).

Figure 5

Effect of non-targeted (DON-SLN) and ApoE-targeted SLNs (APOE-DON-SLN) on the viability of (a) primary rat brain endothelial cells (RBEC) and (b) the human brain endothelial cell line hCMEC/D3 after 2 h of incubation, monitored by impedance measurement. Values presented are means ± SEM and are given as a percentage of control. Statistical analysis: one-way ANOVA followed by Dunnett’s posttest; **** p < 0.0001 compared to the control group; n = 6–8. C: culture medium-treated control group; TX: Triton X-100 treated cells, indicating maximal cellular toxicity.

Figure 6

Effect of non-targeted SLNs (DON-SLN) on the viability of differentiated SH-SY5Y human neuronal cells measured by MTT test. Values presented are means ± SEM and are given as a percentage of control. Statistical analysis: one-way ANOVA followed by Dunnett’s posttest; n = 6–8. C: culture medium-treated control group.

Figure 7

Cellular uptake of rhodamine B (RhB) cargo encapsulated in non-targeted (DON-SLN) and ApoE-targeted SLNs (APOE-DON-SLN) in (a) cultured primary rat brain endothelial cells (RBEC), (b) human brain endothelial cells (hCMEC/D3), and (c) in differentiated SH-SY5Y neuronal cells after 2 h of incubation. Values presented are means ± SEM. Statistical analysis: unpaired t test between DON-SLNs and APOE-DON-SLNs; **** p < 0.0001; n = 6.

Figure 8

Permeability of RhB cargo encapsulated in non-targeted (DON-SLN) or ApoE-targeted SLNs (APOE-DON-SLN) across the BBB model (10 µg/mL concentration, 2 h). Values presented are means ± SEM. Statistical analysis: one-way ANOVA followed by Dunnett’s posttest, where **** p < 0.0001, compared to the DON-SLN group; n = 4. P_app: apparent permeability coefficient, SF: sodium fluorescein (376 Da), EBA: Evans blue-labeled bovine serum albumin (67 kDa).