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

The treatment of neurological disorders (NDs) is challenged by low drug 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. Two-dimensional 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 scale-up 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 (NV-BBB) 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 bio-fabricated 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. This bulk of these 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-related 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 cell-cell communication 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 NV-BBB organoid platform to investigate the BBB permeability of different nanomaterials in nanomedicine, nanosafety, and nanotoxicology.