Multicellular Organoids of the Neurovascular Blood-Brain Barrier: A New Platform for Precision Neuronanomedicine

Abstract

The treatment of neurological disorders (NDs) is challenged by low dug permeability from the systemic circulation into the central nervous system (CNS) owing to the presence of the blood brain barrier (BBB). Neuronanomedicine investigates nanotechnology strategies to target the brain and improve the therapeutic outcome in NDs. 2D adherent cell BBB models show substantial phenogenomic heterogeneity and their ability to predict the permeability of molecules and nanoparticles into the brain is extremely limited. Thus, the high throughput screening of CNS nanomedicines relies on the use of animal models. To address this dearth, 3D organoids that mimic the in vivo physiology are under development. Still, there exist concerns about the standardization and scaleup of the production process, their proper characterisation, and their industrial application. In this work, we report on a novel multicellular organoid of the neurovascular blood brain barrier that recapitulates the regulated syncytium of human endothelial cells and the function of the human BBB. For this, an advanced organoid comprising human brain microvascular endothelial cells, brain vascular pericytes and human astrocytes combined with primary neurons and microglia isolated from neonate rats is biofabricated without the use of an extracellular matrix. The structure and function are fully characterized by confocal laser scanning fluorescence microscopy, light sheet fluorescence microscopy, scanning transmission electron microscopy, cryogenic scanning electron microscopy, western blotting, RNA sequencing and quantitative gene expression by quantitative polymerase chain reaction analysis. The bulk of this self assembloids is comprised of neural cells and microglia and the surface covered by endothelial cells that act as a biological barrier that resembles the BBB endothelium. In addition, the formation of neuron microglia morphofunctional communication sites is confirmed. Analysis of key transcriptomic expressions show the up-regulation of selected BBB genes including tight junction proteins, solute carriers, transporters of the ATP-binding cassette superfamily, metabolic enzymes, and prominent basement membrane signatures. Results confirmed the more efficient intercellular communications in 3D organoids made of multiple neural-tissue cells than in 2D endothelial cell monocultures. These multicellular organoids are utilized to screen the permeability of different polymeric, metallic, and ceramic nanoparticles. Results reveal penetration through different mechanisms such as clathrin mediated endocytosis and distribution patterns in the organoid that depend on the nanoparticle type, highlighting the promise of this simple, reproducible and scalable multicellular organoid platform to investigate the BBB permeability of different nanomaterials in nanomedicine, nanosafety, and nanotoxicology.

The effects of silver nanoparticle shape on protein adsorption and neural stem cell viability

“…Rod-shaped cerium oxide (CeO 2 ) NPs were also more toxic than octahedral or cubic particles in terms of LDH and tumor necrosis factor alpha (TNF) release in macrophages [64]. In a previous work, we demonstrated that, the low accumulation of 60nm sized Ag NPs (60 ± 13 nm) in neurovascular blood-brain barrier (NV-BBB) organoids due to its dissolution outside and inside the brain microenvironment [38,39]. There is therefore increasing data suggesting that the geometric properties of NPs mediate toxicity, but a more systematic approach would further advance our knowledge with respect to exactly which shapes exert the most cytotoxic effects.…”

“…Recent studies have also demonstrated that the geometries of NPs impact protein corona formation [33], circulation time, cellular uptake, and bio-distribution [34]. In the last few years, we conducted systematic in vitro 2-dimensional [35][36][37] and 3-dimensional (brain organoids and spheroids) [38,39] studies to evaluate cellular neurosafety and neural uptake of well-characterized polymeric, metallic (Ag and Au) and ceramic NPs. Among various physico-chemical properties known to in uence biological activities, In this study we focused on the different shape of Ag NPs.…”