Nanoparticles targeting transporters of the blood-brain barrier (BBB) are promising
candidates to increase the brain penetration of biopharmacons. Solute carriers (SLC)
are expressed at high levels in brain endothelial cells and show a specific pattern
at the BBB. The aim of our study was to test glutathione and ligands of SLC transporters
as single or dual BBB targeting molecules for nanovesicles. High mRNA expression levels
for hexose and neutral amino acid transporting SLCs were found in isolated rat brain
microvessels and our rat primary cell based co-culture BBB model. Niosomes were derivatized
with glutathione and SLC ligands glucopyranose and alanine. Serum albumin complexed
with Evans blue (67kDa), which has a very low BBB penetration, was selected as a cargo.
The presence of targeting ligands on niosomes, especially dual labeling, increased
the uptake of the cargo molecule in cultured brain endothelial cells. This cellular
uptake was temperature dependent and could be decreased with a metabolic inhibitor
and endocytosis blockers filipin and cytochalasin D. Making the negative surface charge
of brain endothelial cells more positive with a cationic lipid or digesting the glycocalyx
with neuraminidase elevated the uptake of the cargo after treatment with targeted
nanocarriers. Treatment with niosomes increased plasma membrane fluidity, suggesting
the fusion of nanovesicles with endothelial cell membranes. Targeting ligands elevated
the permeability of the cargo across the BBB in the culture model and in mice, and
dual-ligand decoration of niosomes was more effective than single ligand labeling.
Our data indicate that dual labeling with ligands of multiple SLC transporters can
potentially be exploited for BBB targeting of nanoparticles.