In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Summary

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

Abstract

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

Introduction

The resistance of central nervous system (CNS) diseases (i.e. cancer, epilepsy, depression, schizophrenia and HIV-associated neurological disorder) to pharmacological therapies is due to various different mechanisms, including arduous drug permeation across the blood-brain barrier (BBB). The BBB is the boundary that isolates brain tissues from the substances circulating in the blood. Within this barrier, a layer of brain microvascular endothelial cells (BMECs), supported by pericytes and astrocytes endfeet, is responsible for the high selectivity of the BBB to those hydrosoluble drugs with a molecular weight higher than 400 Da¹. Another drug-relate....

Protocol

1. Establishing the BBB Model

Note: For establishing the BBB model we suggest using commercially available primary RBMECs and RCAs. All steps must be performed with sterile reagents and disposables, handled in a laminar flow hood. 

1. Cell Culture
   1. Coat cell culture flasks with poly-L-lysine 100 µg/ml (1 hr at RT) or fibronectin 50 µg/ml (1 hr at 37 °C) to promote the attachment of RCAs or RBMECs, respectively. Then, thaw 1 x 10⁶ RCAs and 5 x 10⁵ RBMECs in Endothelial Cell Medium, supplemented with 5% fetal bovine serum, 1% endothelial cell growth supplement and 1% penicillin/streptomycin (sECM). See....

Results

During the establishment of the BBB model, cell attachment and growth on the inserts can be monitored using a light microscope thanks to the transparent nature of the PET membranes. RCAs, seeded at a density of 35,000 cells/cm², attach efficiently to the bottom side of the insert after 4 hr of incubation at RT (Figure 2A) and grow to cover the membrane surface in 3 days, taking a spindle-shaped morphology (Figure 2B). RBMECs, seeded at a densit.......

Discussion

The in vitro method described here represents a useful validated approach to study the trans-BBB delivery of fluorescent molecules upon nanoformulation with nanoparticles. Here we use FnN, which represents a good candidate to study the translocation of cargo molecules across the BBB. FnN is considered the gold nanovector for trans-BBB delivery of drug/agents since it is specifically recognized by the TfR1 receptor, which is expressed on the luminal membrane of BMECs and mediates the nanoparticle uptake using a r.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rat Brain Microvascular Endothelial Cells	Innoprot	P10308	isolated from Sprague Dawley rat brain tissue, cryopreserved at passage one and delivered frozen
Cortical Astrocytes	Innoprot	P10202	isolated from 2 days rat brain tissue, cryopreserved at passage one and delivered frozen.
Endothelial Cell Medium kit	Innoprot	P60104	ECM (500 ml) and fetal bovin serum (25 ml), endothelial cell growth supplement (5 ml) and penicillin/streptomycin (5 ml). Warm in 37 °C water bath before use and protect from light
Trypsin-EDTA without Phenol Red	EuroClone	ECM0920D	Warm in 37 °C water bath before use
Fluorescein isothiocyanate-dextran 40,000	Sigma	FD40S	protect from light
paraformaldehyde	Sigma	158127	diluition in chemical hood
Dulbecco's phosphate buffer saline w/o Ca and Mg	EuroClone	ECB4004L
Triton X-100	Sigma	T8787
bovine serum albumin	Sigma	A7906
goat serum	EuroClone	ECS0200D
mouse monoclonal anti-Von Willebrand Factor	Dako	M0616
AlexaFluor 546-conjugated antibody against mouse IgGs	ThermoFischer Scientific	A-11003	protect from light
DAPI (4’ ,6-diamidino-2-phenylindole)	ThermoFischer Scientific	D1306	protect from light
ProLong Gold Antifade Mountant	ThermoFischer Scientific	P36934
Poly-L-lysine Hydrobromide	Sigma	P1274	the same solution can be used several times
fibronectin from bovine plasma	Sigma	F1141	the same solution can be used several times
Polyethylene terephthalate (PET) inserts	Falcon	F3090	Transparent Polyethylene terephthalate (PET) membranes; surface area: 4.2 cm2; pore size 0.4 µm/surface area
T75 Primo TC flask	EuroClone	ET7076
T175 Primo TC flask	EuroClone	ET7181
EVOM2 Epithelial Tissue Volt/Ohmmeter	World Precision Instruments Germany	EVOM2
Endohm- 24SNAP cup	World Precision Instruments Germany	ENDOHM-24SNAP
Light/fluorescence microscope with camera	Leica Microsystems	DM IL LED Fluo/ ICC50 W Camera Module	inverted microscope for live cells with camera
Confocal Microscope	Leica Microsystems	TCS SPE
Spectrofluorimeter	Jasco	FP-8000