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. Etablering BBB Model Bemærk: oprettelse af BBB model, vi foreslår at bruge kommercielt tilgængelige primære RBMECs og RCAS. Alle trin skal udføres med sterile reagenser og engangsartikler, der håndteres i en laminar flow hætte. <ol><li> celledyrkning <ol><li> Coat celle kultur flasker med poly-L-lysin 100 ug / ml (1 time ved stuetemperatur) eller fibronectin 50 ug / ml (1 time ved 37 ° C) for at fremme fastgørelsen af ​​RCAS eller RBMECs hhv. Derefter tø 1 x 10 6 RCAS og 5 x 10 <sup...

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In vitro-metoden beskrevet her repræsenterer en nyttig valideret metode til at studere trans-BBB levering af fluorescerende molekyler ved nanoformulation med nanopartikler. Her bruger vi Fnn, som repræsenterer en god kandidat til at studere translokationen af ​​last molekyler over BBB. Fnn anses guld nanovector for trans-BBB levering af narkotika / agenter, da det specifikt er anerkendt af TfR1 receptoren, som udtrykkes på den luminale membran af BMECs og medierer nanopartikel optagelse ved hjælp af en ...

Modstanden i centralnervesystemet (CNS) sygdomme (dvs.. Kræft, epilepsi, depression, skizofreni og HIV-associeret neurologisk lidelse) til farmakologiske behandlinger skyldes forskellige mekanismer, herunder besværlig lægemiddel gennemtrængning af blod-hjerne-barrieren (BBB) . BBB er grænsen, som isolerer hjernevæv fra stofferne cirkulerer i blodet. Inden for denne barriere, et lag af hjerne mikrovaskulære endotelceller (BMECs), støttet af pericytter og astrocytter endfeet, er ansvarlig for den høje selektivitet af BBB til disse vandopløselige lægemidler med en molekylvægt højere end 400 Da 1. En anden narkotikarelaterede modstand mekanisme er forbundet med tilstedeværelsen på BMECs af narkotika effluxtransportører (P-glycoprotein og multiresistens proteiner), som co-driver for at reducere indtrængning lægemiddel i CNS og lette deres ekstrudering fra hjernen 2. I det sidste årti, Et stort antal nanoteknologiske fremgangsmåder er blevet udviklet til at opfylde den kliniske og biologiske udfordring at levere lægemidler over BBB 3-6. I denne sammenhæng, ferritin nanosfærer (Fnn) repræsenterer en helt nyskabende og lovende løsning. Fnn er 12 nm kugler af 24 selvsamlende ferritin (Fn) monomerer, som er anbragt i en hul sfærisk struktur 8 nm indre diameter. Ferritin subunits kan skilles ad ved sur pH og samles igen i et SME-mode ved at bringe pH-værdien til neutralitet, hvilket giver forskellige organiske molekyler, der skal indkapsles. Derfor Fnn repræsenterer en interessant model for udvikling af multifunktionelle lægemiddelafgivelsessystemer 7,8. Endvidere kan Fnn interagere med BMECs takket være specifik genkendelse af transferrinreceptoren (TfR) 1, som udtrykkes på den luminale membran af disse celler 9. Hidtil anderledes in vitro modeller af BBB er udviklet in order for at belyse trans-BBB gennemtrængelighed for forskellige stoffer, toksicitet mod BBB, eller interaktionen af ​​molekyler med effluxtransportører. Faktisk er disse modeller anses for gyldig i vitro tilgange til en hurtig screening af aktive molekyler, før du fortsætter med in vivo studier. Disse modeller består af et enkelt endotel lag BMECs eller co-dyrkede BMECs og astrocytter (mere sjældent pericytes), fremstillet af dyr (rotte, mus, svin og kvæg) eller humane cellelinjer 10,11,12. Den transendotelial elektrisk modstand (TEER) og den tilsyneladende permeabilitet (Papp) af sporstoffer med en defineret molekylvægt repræsenterer to kritiske parametre, der anvendes til at bestemme kvaliteten af den in vitro model. Her beskriver vi ansættelse af en BBB in vitro-model, der bygger på en co-kultur af rotte BMECs (RBMECs) og rotte kortikale astrocytter (RCAS) at undersøge den trans-BBB gennemtrængning af ferritin nanocages indkapsle 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.

Under etableringen af ​​BBB-modellen, cellebinding og vækst på indsatsene kan overvåges ved hjælp af et lysmikroskop takket være den åbne karakter PET membraner. RCAS, podet med en tæthed på 35.000 celler / cm2, vedhæfte effektivt til undersiden af indsatsen efter 4 timer inkubation ved RT (figur 2A) og vokse til at dække membranoverfladen i 3 dage under en spindel-formet morfologi (Figur 2B). RBMECs, podet ved en densitet på 60...

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 gennemtrængning af FITC-loaded Ferritins tværs af en rotte blodhjernebarrieren: en model til at studere den Levering af Nanoformulated molekyler

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

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

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