Internal benchmarking of a human blood–brain barrier cell model for screening of nanoparticle uptake and transcytosis

Ragnaill, Michelle Nic; Brown, Meredith S.; Ye, Dong; Bramini, Mattia; Callanan, John J.; Lynch, Iseult; Dawson, Kenneth A.

doi:10.1016/j.ejpb.2010.12.024

Cited by 80 publications

(72 citation statements)

References 18 publications

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“…S1) by culturing the cells on filters until confluency and tight junction formation (typically after 7-10 days), as assessed by tight junction protein staining, TEER value measurements (40-50 Ω•cm 2 ) and FITC dextran permeability studies, as previously described. 46 In some cases, endothelial cells formed multiple layers on the transwell filters, rather than a monolayer (See Supplementary Fig. S2 for some examples), and this could signal the presence of areas which were not properly differentiated.…”

Section: Resultsmentioning

confidence: 99%

“…By analyzing the ratio of a compound travelling through the model, we can rank the transport efficiency of different compounds. 40,46 However, we have observed several limitations which hinder the study of nanoparticle transport through the endothelial cells in such transwell systems.…”

Section: Resultsmentioning

confidence: 99%

“…44,45 In this work, we studied the fate of various types of nano-sized materials accessing the blood brain barrier, and we used the hCMEC/D3 cells as an in vitro BBB model for nanoparticle screening. 46 Unmodified bare silica (SiO 2 ) nanoparticles of different sizes, anatase titanium dioxide (TiO 2 ) nanoparticles and gold (Au) nanoparticles of different sizes, conjugated with endogenous proteins (albumin or transferrin) were applied to this model in typical in vitro concentrations (10-100 47 Fluorescence based methods can be efficiently used to quantify the uptake of fluorescently labeled nanoparticles by flow cytometry and/or fluorescence imaging and also to study nanoparticle subcellular locations. 48,49 However, we found that for nanoparticle applications, classic transport studies by fluorescence using transwell systems presents several limits, and in some cases can produce unreliable results, due to technical limitations of nanoparticle fluorescence and nanoparticle adherence to the transwell filter pores.…”

Section: Introductionmentioning

confidence: 99%

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Nanoparticle accumulation and transcytosis in brain endothelial cell layers

et al. 2013

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The Blood-Brain Barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight junctions, which limit paracellular permeability. Several reports suggest that nanoparticles have a unique capacity to cross the BBB. However, direct evidence of nanoparticle transcytosis is difficult to obtain, and we found that typical transport studies present several limitations when applied to nanoparticles. In order to investigate the capacity of nanoparticles to access and transport across the BBB, several different nanomaterials, including silica, titania and albumin-or transferrinconjugated gold nanoparticles of different sizes, were exposed to a human in vitro BBB model of endothelial hCMEC/D3 cells. Extensive transmission electron microscopy imaging was applied in order to describe nanoparticle endocytosis and typical intracellular localisation, as well as to look for evidence of eventual transcytosis. Our results show that all of the nanoparticles were internalised, to different extents, by the BBB model and accumulated along the endo-lysosomal pathway. Rare events suggestive of nanoparticle transcytosis were also observed for several of the tested materials.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Nanoparticle accumulation and transcytosis in brain endothelial cell layers

et al. 2013

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“…We have previously demonstrated that this model can be used to study NP interactions with, uptake into and transport across the BBB. 29 Our findings then, and on the basis of significant additional imaging work, suggested that only very small numbers of NPs cross the in vitro BBB. Building on that, the present paper investigates the effect of NP accumulation in the BBB on lysosome health and paracrine signalling, as it is clear that impacts on the BBB cells could have dramatic consequences for the vulnerable tissues protected by biological barriers.…”

Section: Introductionmentioning

confidence: 61%

Paracrine signalling of inflammatory cytokines from an in vitro blood brain barrier model upon exposure to polymeric nanoparticles

Raghnaill

Bramini

Dong

et al. 2014

Analyst

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Nanoparticle properties, such as small size relative to large highly modifiable surface area, offer great promise for neuro-therapeutics and nanodiagnostics. A fundamental understanding and control of how nanoparticles interact with the blood-brain barrier (BBB) could enable major developments in nanomedical treatment of previously intractable neurological disorders, and help ensure that nanoparticles not intended to reach the brain do not cause adverse effects. Nanosafety is of utmost importance to this field. However, a distinct lack of knowledge exists regarding nanoparticle accumulation within the BBB and the biological effects this may induce on neighbouring cells of the Central Nervous System (CNS), particularly in the long-term. This study focussed on the exposure of an in vitro BBB model to model carboxylated polystyrene nanoparticles (PS COOH NPs), as these nanoparticles are well characterised for in vitro experimentation and have been reported as non-toxic in many biological settings. TEM imaging showed accumulation but not degradation of 100 nm PS COOH NPs within the lysosomes of the in vitro BBB over time. Cytokine secretion analysis from the in vitro BBB post 24 h 100 nm PS COOH NP exposure showed a low level of proinflammatory RANTES protein secretion compared to control. In contrast, 24 h exposure of the in vitro BBB endothelium to 100 nm PS COOH NPs in the presence of underlying astrocytes caused a significant increase in pro-survival signalling. In conclusion, the tantalising possibilities of nanomedicine must be balanced by cautious studies into the possible long-term toxicity caused by accumulation of known 'toxic' and 'non-toxic' nanoparticles, as general toxicity assays may be disguising significant signalling regulation during long-term accumulation.

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“…The combination of transmission electron microscopy (TEM) with traditional Transwell ™ systems has revealed some crucial limitations of these models (18,44,45), which bring into question the reliability and reproducibility of these systems. These limitations include barrier imperfections, such as formation of multiple layers of endothelial cells on the filter and holes (see Figure 3); nanoparticle adhesion to the microporous filter pore; nanoparticle agglomeration in the culture medium and inside the cell; and leakage of the fluorescent dye.…”

Section: Investigation Of Transcytosis and Other Mechanisms Through Tmentioning

confidence: 99%

Reproducibility in biological models of the blood-brain barrier

Hudecz

Rocks

Fitzpatrick

et al. 2014

European Journal of Nanomedicine

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Abstract:In the past decade, the importance of quality and reproducibility within research has been re-emphasized, consequently becoming a crucial part of scientific experiments. Their implementation into in vitro and in vivo biological experiments is challenging due to various parameters that can influence the final scientific outcome. In parallel to these activities, there is a huge scientific effort to improve today's medicines to make them safer and more efficient, and to cure untreatable diseases, such as many neurodegenerative diseases. Nanosized materials have been recognized as potential drug delivery systems in this arena due to their small size and surface properties, which enable the design and synthesis of safe and efficient delivery vehicles that might be able to cross the bloodbrain barrier. However, the fundamental understanding behind their uptake mechanism and intracellular trafficking remains unknown. Simple and cost-effective in vitro blood-brain barrier models are widely used to address these questions. This paper aims to critically evaluate the current in vitro models using Transwell ™ systems and to discuss alternative approaches towards more reproducible in vivo features.

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Internal benchmarking of a human blood–brain barrier cell model for screening of nanoparticle uptake and transcytosis

Cited by 80 publications

References 18 publications

Nanoparticle accumulation and transcytosis in brain endothelial cell layers

Nanoparticle accumulation and transcytosis in brain endothelial cell layers

Paracrine signalling of inflammatory cytokines from an in vitro blood brain barrier model upon exposure to polymeric nanoparticles

Reproducibility in biological models of the blood-brain barrier

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