title: A methodology to study intracellular distribution of nanoparticles in brain endothelial cells.
abstract: Cell internalisation and intracellular distribution of PEG-coated polyhexadecylcyanoacrylate (PEG-PHDCA) nanoparticles in rat brain endothelial cells (RBEC) have been investigated. A cell fractionation method has been developed based on the selective permeabilisation of RBEC plasma membrane by digitonin. By interacting with membrane cholesterol, digitonin creates pores allowing the release of soluble and diffusible species outside the cell. The selectivity of plasma membrane permeabilisation was controlled by using compartment markers such as lactate dehydrogenase (LDH) for cytoplasm and cathepsin B for lysosomes. An optimal digitonin concentration of 0.003% (w/v) has been identified to induce a pattern of membrane permeabilisation corresponding to the extraction of 72% LDH and less than 15% of Cathepsin B. Membrane permeabilisation at this digitonin concentration allows one to distinguish between the cell cytoplasm and its endo/lysosomal fraction. This methodology was applied to investigate the intracellular distribution of the nanoparticles after their incubation with the RBEC. The results showed that PEG-PHDCA nanoparticles were able to be internalised to a higher extent than PHDCA nanoparticles (after 20 min incubation). Additionally, these nanoparticles displayed different patterns of intracellular capture, depending on their specific surface composition: PEG-PHDCA nanoparticles were 48% in the plasma membrane, 24% in the cytoplasm, 20% in vesicular compartments and 8% associated with the fraction of the nucleus, the cytoskeleton and caveolae suggesting that PEG-PHDCA nanoparticle uptake by RBEC is specific and presumably due to endocytosis. Confocal microscopy studies confirmed the cellular uptake of PEG-PHDCA nanoparticles.
authors.affiliation: Laboratory of Biopharmacy and Pharmaceutical Technology, UMR CNRS 8612, Faculty of Pharmacy, University of Paris-XI, 92296 Châtenay-Malabry, France.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Garcia-Garcia
authors.forename: Karine
authors.initials: K
authors.lastname: Andrieux
authors.forename: Sophie
authors.initials: S
authors.lastname: Gil
authors.forename: Hyun Ryoung
authors.initials: HR
authors.lastname: Kim
authors.forename: Trung
authors.initials: T
authors.lastname: Le Doan
authors.forename: Didier
authors.initials: D
authors.lastname: Desmaële
authors.forename: Jean
authors.initials: J
authors.lastname: d'Angelo
authors.forename: Frédéric
authors.initials: F
authors.lastname: Taran
authors.forename: Dominique
authors.initials: D
authors.lastname: Georgin
authors.forename: Patrick
authors.initials: P
authors.lastname: Couvreur
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology*; D001923:Brain Chemistry / Q000502:physiology*; D002401:Cathepsin B / Q000494:pharmacology; D002458:Cell Fractionation; D016503:Drug Delivery Systems; D042783:Endothelial Cells / Q000378:metabolism*; D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase / Q000494:pharmacology; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D049329:Nanostructures; D010447:Peptide Hydrolases / Q000737:chemistry; D051381:Rats
chemical_list: D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase; D010447:Peptide Hydrolases; D002401:Cathepsin B