Name: in vitro permeation of fitc-loaded ferritins across a rat blood-brain barrier: a model to study the delivery of nanoformulated molecules
Uploaded: 2016-08-22T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules at JoVE.com

The authors acknowledge Assessorato alla Sanit&#224;, Regione Lombardia and Sacco Hospital (NanoMeDia Project) for research funding.

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.&#160;
<ol>
	<li>Cell Culture
	<ol>
		<li>Coat cell culture flasks with poly-L-lysine 100 &#181;g/ml (1 hr at RT) or fibronectin 50 &#181;g/ml (1 hr at 37 &#176;C) to promote the attachment of RCAs or RBMECs, respectively. Then, thaw 1 x 106 RCAs and 5 x 105 RBMECs...

<ol>
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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...

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-related resistance mechanism is linked to the presence on BMECs of drug efflux transporters (P-glycoprotein and multidrug resistance proteins), which co-operate to reduce drug penetration into the CNS and facilitate their extrusion from the brain2.
In the last decade, a large number of nanotechnological approaches have been developed to meet the clinical and biological challenge of delivering drugs across the BBB3-6. In this context, ferritin nanospheres (FnN) represent a completely innovative and promising solution. FnN are 12 nm spheres of 24 self-assembling ferritin (Fn) monomers, which are arranged in a hollow spherical structure of 8 nm inner diameter. Ferritin subunits can be disassembled at acidic pH and reassembled in a shape-memory fashion by bringing the pH to neutrality, allowing various organic molecules to be encapsulated. Therefore, FnN represent an interesting model for the development of multifunctional drug delivery systems7,8. Moreover, FnN may interact with BMECs thanks to the specific recognition of Transferrin Receptor (TfR) 1, which is expressed on the luminal membrane of these cells9.
So far, different in vitro models of the BBB have been developed in order to elucidate trans-BBB permeability to various drugs, toxicity toward the BBB, or the interaction of molecules with efflux transporters. Indeed, these models are considered valid in vitro approaches for a rapid screening of active molecules before proceeding with in vivo studies. These models consist of a single endothelial layer of BMECs or co-cultured BMECs and astrocytes (more rarely pericytes), obtained from animal (rat, mouse, pig and bovine) or human cell lines10,11,12. The TransEndothelial Electrical Resistance (TEER) and the apparent permeability (Papp) of tracers with a defined molecular weight represent two critical parameters that are used to determine the quality of the in vitro model. Here we describe the employment of a BBB in vitro model, based on a co-culture of rat BMECs (RBMECs) and rat cortical astrocytes (RCAs) to study the trans-BBB permeation of ferritin nanocages encapsulating fluorescein isothiocyanate (FITC).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Rat Brain Microvascular Endothelial Cells&#160;</td>
			<td>Innoprot</td>
			<td>P10308</td>
			<td>isolated from Sprague Dawley rat brain tissue, cryopreserved at passage one and delivered frozen</td>
		</tr>
		<tr>
			<td>Cortical Astrocytes&#160;</td>
			<td>Innoprot</td>
			<td>P10202</td>
			<td>isolated from 2 days rat brain tissue, cryopreserved at passage one and delivered frozen.</td>
		</tr>
		<tr>
			<td>Endothelial Cell Medium kit</td>
			<td>Innoprot</td>
			<td>P60104</td>
			<td>ECM (500 ml) and fetal bovin serum (25 ml), endothelial cell growth supplement (5 ml) and penicillin/streptomycin (5 ml). Warm in 37 &#176;C water bath before use and protect from light</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA without Phenol Red</td>
			<td>EuroClone</td>
			<td>ECM0920D</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate-dextran 40000</td>
			<td>Sigma</td>
			<td>FD40S</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>paraformaldehyde&#160;</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>diluition in chemical hood</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffer saline w/o Ca and Mg</td>
			<td>EuroClone</td>
			<td>ECB4004L</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin&#160;</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>goat serum&#160;</td>
			<td>EuroClone</td>
			<td>ECS0200D</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-Von Willebrand Factor</td>
			<td>Dako</td>
			<td>M0616</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 546-conjugated antibody against mouse IgGs</td>
			<td>ThermoFischer Scientific</td>
			<td>A-11003</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>DAPI (4&#8217; ,6-diamidino-2-phenylindole)&#160;</td>
			<td>ThermoFischer Scientific</td>
			<td>D1306</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFischer Scientific</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Hydrobromide</td>
			<td>Sigma</td>
			<td>P1274</td>
			<td>the same solution can be used several times</td>
		</tr>
		<tr>
			<td>fibronectin from bovine plasma&#160;</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td>the same solution can be used several times</td>
		</tr>
		<tr>
			<td>Polyethylene terephthalate (PET) inserts</td>
			<td>Falcon</td>
			<td>F3090</td>
			<td>Transparent Polyethylene terephthalate (PET) membranes; surface area: 4.2 cm2; pore size 0.4 &#181;m/surface area</td>
		</tr>
		<tr>
			<td>T75 Primo TC flask</td>
			<td>EuroClone</td>
			<td>ET7076</td>
			<td></td>
		</tr>
		<tr>
			<td>T175 Primo TC flask</td>
			<td>EuroClone</td>
			<td>ET7181</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2 Epithelial Tissue Volt/Ohmmeter&#160;&#160;&#160;</td>
			<td>World Precision Instruments Germany</td>
			<td>EVOM2</td>
			<td></td>
		</tr>
		<tr>
			<td>Endohm- 24SNAP cup</td>
			<td>World Precision Instruments Germany</td>
			<td>ENDOHM-24SNAP</td>
			<td></td>
		</tr>
		<tr>
			<td>Light/fluorescence microscope with camera</td>
			<td>Leica Microsystems</td>
			<td>DM IL LED Fluo/ ICC50 W Camera Module</td>
			<td>&#160;inverted microscope for live cells with camera&#160;</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica Microsystems</td>
			<td>TCS SPE</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro permeation of fitc-loaded ferritins across a rat blood-brain barrier: a model to study the delivery of nanoformulated 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.

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...

Watch this Scientific Journal Video about In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules at JoVE.com

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

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.

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

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

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