BBB-on-a-chip with Integrated micro-TEER for permeability evaluation of multi-functionalized gold nanorods against Alzheimer’s disease

Abstract

Background The lack of predictive models that mimic the blood-brain barrier (BBB) hinders the development of effective drugs for neurodegenerative diseases. Animal models behave differently from humans, are expensive and have ethical constraints. Organ-on-a-chip (OoC) platforms offer several advantages to resembling physiological and pathological conditions in a versatile, reproducible, and animal-free manner. In addition, OoC give us the possibility to incorporate sensors to determine cell culture features such as trans-endothelial electrical resistance (TEER). Here, we developed a BBB-on-a-chip (BBB-oC) platform with a TEER measurement system in close distance to the barrier used for the first time for the evaluation of the permeability performance of targeted gold nanorods for theranostics of Alzheimer's disease. GNR-PEG-Ang2/D1 is a therapeutic nanosystem previously developed by us consisting of gold nanorods (GNR) functionalized with polyethylene glycol (PEG), angiopep-2 peptide (Ang2) to overcome the BBB and the D1 peptide as beta amyloid fibrillation inhibitor, finally obtaining GNR-PEG-Ang2/D1 which showed to be useful for disaggregation of the amyloid in in vitro and in vivo models. In this work, we evaluated its cytotoxicity, permeability, and some indications of its impact on the brain endothelium by employing an animal-free device based on neurovascular human cells. Results In this work, we fabricated a BBB-oC with human astrocytes, pericytes and endothelial cells and a TEER measuring system (TEER-BBB-oC) integrated at a micrometric distance of the endothelial barrier. The characterization displayed a neurovascular network and the expression of tight junctions in the endothelium. We produced GNR-PEG-Ang2/D1 and determined its non-cytotoxic range (0.05–0.4 nM) for plated cells included in the BBB-oC and confirmed its harmless effect at the highest concentration (0.4 nM) in the microfluidic device. The permeability assays revealed that GNR-PEG-Ang2/D1 cross the BBB and this entry is facilitated by Ang2 peptide. Parallel to the permeability analysis of GNR-PEG-Ang2/D1, an interesting behavior of the TJs expression was observed after its administration probably related to the ligands on the nanoparticle surface. Conclusion BBB-oC with a novel TEER integrated setup which allow a correct read-out and cell imaging monitoring was proven as a functional and throughput platform to evaluate the brain permeability performance of nanotherapeutics in a physiological environment with human cells, putting forward a viable alternative to animal experimentation.

Lab-on-a-chip models of the blood–brain barrier: evolution, problems, perspectives

“…40 In addition to successful imaging of fluorescently labelled HDL particles, the study also conducted diffusion analysis and single molecule tracking. The results proved that although HDL particles interact with the luminal surface of brain This journal is © The Royal Society of Chemistry 2024 Table 6 BBB-on-a-chip systems for nanoparticle penetration 22,32,40,47,50,51,54,57,59,63 endothelial cells they do not pass across the in vitro BBB coculture model. 40 Transferrin receptors are also highly expressed at the BBB, and transferrin, its peptide sequences or antibodies specific for transferrin receptor are widely used to target nanoparticles to brain.…”

“…Ideally, non-targeted and functionalized nanocarriers are compared in the same setting (Table 6 ). 22,32,40,47,50,51,54,57,59,63 The most investigated targeting ligand for nanoparticles in BBB-on-chip systems was the angiopep-2 peptide targeting low density lipoprotein receptor-related proteins (LRPs) abundantly expressed at the BBB both in vivo [113][114][115] and in culture models. 14,56,87 The penetration of liposomes functionalized with angiopep-2 peptide, was demonstrated across bEnd.3 mouse brain endothelial cell monolayers cultured in the upper compartment of a 2-channel microfluidic device.…”

“…59 Angiopep-2 also facilitated the permeability of gold nanorods designed for theranostic application. 54 The BBB model was a hydrogel-based self-organized microvascular network in a 2-channel microfluidic chip equipped with TEER measurement system. The permeability properties of this BBB model for fluorescein and dextran markers are well fitting literature data (Table 6).…”

Patient‐Derived Microphysiological Systems for Precision Medicine

“…[52,225] Barrier models like the BBB can guide the generation of standardized data from MPS equipped with sensors like TEER around the barrier. [226,227] Researchers can collaboratively develop and share assay methods, incorporating real-time/functional data collection from P-MPS and integrating sensors within the device. [66,228] Third, adopting AI-driven processing for swift and reliable data generation is crucial, as previously discussed.…”

Sensor‐integrated brain‐on‐a‐chip platforms: Improving the predictive validity in neurodegenerative research

“…Representative examples of TEER‐integrated OoC platforms using either gold‐sputtered (b, c) or inserted Ag/AgCl electrodes (d). Reproduced with permission from Henry et al, 88 2017, The Royal Society of Chemistry (b), Palma‐Florez et al, 95 2023, Springer Nature (c), and Zoio et al, 90 2021, MDPI (d). Graphical representation of electrical cell‐substrate impedance sensing (ECIS) measurements (e).…”