Fiandra Luisa, Mazzucchelli Serena, Truffi Marta, Bellini Michela, Sorrentino Luca, Corsi Fabio
Dipartimento di Scienze Biomediche e Cliniche Luigi Sacco, Università di Milano;
Dipartimento di Scienze Biomediche e Cliniche Luigi Sacco, Università di Milano.
J Vis Exp. 2016 Aug 22(114):54279. doi: 10.3791/54279.
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.
脑微血管内皮细胞在周细胞和星形胶质细胞终足的支持下,导致大分子水溶性药物难以透过血脑屏障(BBB),给有效的药物治疗带来困难。近年来,不同的促进脑靶向的策略旨在改善药物在该部位的递送和活性,包括用于跨血脑屏障药物递送的创新纳米系统。在此背景下,基于血脑屏障简化细胞模型的体外方法为研究纳米制剂对分子跨血脑屏障渗透的影响提供了有用工具。本研究描述了一种双层血脑屏障的构建,其由共培养的市售原代大鼠脑微血管内皮细胞和星形胶质细胞组成。还描述了一种基于跨内皮电阻测量和高分子量葡聚糖表观渗透率的多参数模型验证方法。作为使用该血脑屏障模型研究不同纳米制剂对荧光分子跨屏障转运影响的概念验证,我们描述了装载到铁蛋白纳米颗粒中的异硫氰酸荧光素(FITC)的应用。通过通量测量和共聚焦显微镜分析证明了铁蛋白改善FITC跨血脑屏障渗透的能力。结果表明,这是一个用于验证跨血脑屏障药物递送纳米系统的有用系统。