In Vitro Blood-Brain Barrier Models for Nanomedicine: Particle-Specific Effects and Methodological Drawbacks.

Predicting the therapeutic efficacy of a nanocarrier, in a rapid and cost-effective way, is pivotal for the drug delivery to the central nervous system (CNS). In this context, in vitro testing platforms, like the transwell systems, offer numerous advantages to study the passage through the blood-brain barrier (BBB), such as overcoming ethical and methodological issues of in vivo models. However, the use of different transwell filters and nanocarriers with various physical-chemical features makes it difficult to assess the nanocarrier efficacy and achieve data reproducibility. In this work, we performed a systematic study to elucidate the role of the most widely used transwell filters in affecting the passage of nanocarriers, as a function of filter pore size and density. In particular, the transport of carboxyl- and amine-modified 100 nm polystyrene nanoparticles (NPs), chosen as model nanocarriers, was quantified and compared to the behavior of Lucifer yellow (LY), a molecular marker of paracellular transport. Results indicate that the filter type affects the growth and formation of the confluent endothelial barrier, as well as the transport of NPs. Interestingly, the in situ dispersion of NPs was found to play a key role in governing their passage through the filters, both in absence and in presence of the cellular barrier. By framing the underlying nanobiointeractions, we found that particle-specific effects modulated cellular uptake and barrier intracellular distribution, eventually governing transcytosis through their interplay with "size exclusion effects" by the porous filters. This study highlights the importance of a careful evaluation of the physical-chemical profile of the tested nanocarrier along with filter parameters for a correct methodological approach to test BBB permeability in nanomedicine.