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Bioengineering

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

Published: August 22nd, 2016

DOI:

10.3791/54279

1Dipartimento di Scienze Biomediche e Cliniche Luigi Sacco, Università di Milano, 2Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca

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.

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.

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....

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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.......

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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.......

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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.......

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The authors acknowledge Assessorato alla Sanità, Regione Lombardia and Sacco Hospital (NanoMeDia Project) for research funding.

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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 40000 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

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