A relevant in vitro rat model for the evaluation of blood-brain barrier translocation of nanoparticles

Abstract

Poly(MePEG2000cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to reach the rat central nervous system after intravenous injection. For insight into the transport of colloidal systems across the blood-brain barrier (BBB), we developed a relevant in vitro rat BBB model consisting of a coculture of rat brain endothelial cells (RBECs) and rat astrocytes. The RBECs used in our model displayed and retained structural characteristics of brain endothelial cells, such as expression of P-glycoprotein, occludin and ZO-1, and immunofluorescence studies showed the specific localization of occludin and ZO1. The high values of transendothelial electrical resistance and low permeability coefficients of marker molecules demonstrated the functionality of this model. The comparative passage of polyhexadecylcyanoacrylate and PEG-PHDCA nanoparticles through this model was investigated, showing a higher passage of PEGylated nanoparticles, presumably by endocytosis. This result was confirmed by confocal microscopy. Thanks to a good in vitro/in vivo correlation, this rat BBB model will help in understanding the mechanisms of nanoparticle translocation and in designing new types of colloidal carriers as brain delivery systems.

Figure 1

Characteristics of cultured rat brain endothelial cells (a) and astrocytes (b) by phase-contrast microscopy and immunocytochemistry labeling: (1) unlabeled cells, (2) labeled cells with no specific primary antibodies, (3) labeled cells with specific primary antibodies-von Willebrand factor in cultured RBECs (a3) and GFAP in astrocytes (b3).

Figure 2

a Western blot analysis of occludin, ZO-1 and P-gp in RBECs after 13 days in a coculture system. (b, c) Confocal microscopy of the distribution of occludin, ZO-1 and F-actin in RBECs (b) and HUVEC (c) after 13 days in coculture. F-actin was stained with rhodamine-phalloidin. Bar, 10 µm.

Figure 3

a TEER of endothelial cell monolayers in monoculture (closed circles) and coculture with astrocytes (open circles) for 3 weeks after plating onto a collagen-coated filter insert. All data are expressed as the means (n=15) ± SE. b. Western Blot analysis of occludin in RBECs in the coculture system at 13 days (lane 1), and at 28 days (lane 2); monoculture system at 13 days (lane 3) and at 28 days (lane 4).

Figure 4

Translocation of radiolabeled PHDCA nanoparticles (open triangles), PEG-PHDCA nanoparticles (closed triangles) and degraded PEG-PHDCA nanoparticles (open squares) across cell-free collagen-coated filter inserts (a) and across RBECs after 13 days in coculture with astrocytes (b).

Figure 5

Confocal microscopic images of uptake of fluorescent nanoparticles composed of PHDCA (a) and PEG-PHDCA (b) by rat brain endothelial cells (13 days of culture) after 1 h of incubation.

Figure 6

TEER values of cocultures (13 days of culture) without incubation of nanoparticles (open bars), with incubation of PEG-PHDCA nanoparticles (closed bars) or with incubation of PHDCA nanoparticles (hatched bars). All data are expressed as the means (n=10) ± SE.