The blood-brain barrier (BBB) is among the most important physiological challenges to the successful pharmacological treatment of many neurological conditions. It consists of a specialized vascular boundary shutting off the central nervous system (CNS) from the free traffic of substances in the peripheral circulation.
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The BBB is made up of a network of specialized star-shaped brain cells called astrocytes that form a network around brain microvascular endothelial cells (BMVECs) and versatile cells called pericytes. Astrocytes have end-feet, structures that come in contact with the blood vessels and thus regulate the entrance of water via aquaporin-4 (AQP4). The pericytes are anchored to the vascular basement membrane, wrapping themselves around the endothelium and keeping the astrocytes in the right orientation.
This configuration is essential to creating the tight barrier generated by the endothelial cells.
While the BBB is highly protective against the excessive influx of essential nutrients and metabolites into the brain, it is also frustrating when it comes to treating brain cancers or degenerating neurons. Sometimes, the BBB itself is damaged in spots, toxic metabolites, inflammatory cells, or pathogens to enter the brain.
Many delivery systems have been researched over the years to cross this obstacle. High-density lipoprotein (HDL)-like nanoparticles (NPs) have been used as drug vehicles, with the ligands being specific to the surface of the endothelial cell receptors on the BBB.
These require to be evaluated using accurate experimental models. Species differences often mean that animal models do not provide correct predictions of the drug’s activity in humans, while the complexity of animal physiology makes it hard to predict and uncover the mechanism of action of NPs in real-time.
The recent advent of organ-on-a-chip technology now promises to provide a way out. It uses microfluidic technology to overcome the development of a highly controlled cellular and extracellular matrix but still allows responses to various stimuli.
These systems mimic the complex cellular interactions and structures found in many tissues or organs in vivo, and are thus referred to as 'organ-on-a-chip' “
Even so, there are difficulties in fitting the complex physiology of astrocytes into in vitro models of the human BBB. There is a need to be able to introduce damaged as well as healthy astrocytes into the model and to recreate the physical and physiological functions of the BBB with accuracy.
Dedicated work by several teams at different centers of research has resulted in the development of micro-engineered BBB systems on a chip.
A team from Emory State University created a human BBB-on-chip model with a specialized brain endothelial monolayer and an astrocyte network in a 3D structure. It displays a high expression of BBB-specific proteins, leading to tight barrier and low permeability characteristics.
Several other papers report the successful development of a human BBB-on-a-chip, such as one on a high-throughput plate-based format ideal for drug screening, with high barrier function and differential antibody entry based on the presence of specific receptors.
In one model from Harvard’s Wyss Institute for Biologically Inspired Engineering, the basis is the use of human pluripotent stem (iPS) cells, which are induced to differentiate into brain microvascular endothelial cells (BMVECs) in a hypoxic environment. This model is an extension of already existing microfluidic Organs-on-Chips (Organ Chips) technology, combining it with a new “hypoxia-mimicking” technique.
Why the hypoxia-mimicking model? The authors explain that this was inspired by the development of the BBB during human embryonic life under hypoxic conditions. Accordingly, the iPS cells were differentiated for long periods under 5% oxygen rather than the typical 20% concentration.
The result is a high-fidelity BBB chip that has accurate cellular organization and tight barrier functions, as well as transport functions. Its ability to traffic therapeutic drugs and antibodies is a better reflection of actual BBB transport than earlier in vitro models.
Our approach to modeling drug and antibody shuttling across the human BBB in vitro with such high and unprecedented fidelity presents a significant advance over existing capabilities in this enormously challenging research area.”
Using experience from their past BBB model, the BMVECs were transferred into one channel of a dual-channel microfluidic Organ Chip device, that is filled with a stream of the medium. A porous membrane divides the channels.
The other channel contained astrocytes and pericytes. The chip was then treated with hypoxia for one more day.
Gratifyingly, this led to the sustained development of the BBB chip for two weeks or more under normal conditions. This has already surpassed the maintenance duration of any past in vitro human BBB model.
With continuing development, the BMVECs developed into a blood vessel, while also aligning with pericytes on the other side of the porous membrane to form a strong interface. The astrocytes also extend end-feet through the membrane openings towards the BMVECs.
This pattern of development occurs under the stimulus of the fluid shear stress, and the tight barrier formed in this fashion is accompanied by increased numbers of selective transport systems, as well as drug transport shuttles. In this, it was superior to both BBB chips generated without such shear stress or hypoxia, and to adult brain endothelial cells.
Advantages of human BBB-on-a-chip
Such chips can be used to study drug delivery to the brain, as shown by the results of several experiments that looked into transport mechanisms that inhibit drug entry into the brain, as by promoting their efflux into the peripheral circulation, or conversely, facilitate drug and nutrient transport via selective channels across the BBB, in a process called transcytosis.
In the former category, this chip was manipulated to enhance the entry of the drug called doxorubicin, an anticancer molecule, across the BBB by inhibiting the endothelial efflux pump molecule called P-gp. The results were close to those observed in human patients, indicating the value of the new in vitro system to predict useful approaches in this treatment strategy.
Receptor-mediated transcytosis is another area of focus, which could be used to carry NPs loaded with drug cargo, or bigger particles like protein or chemical compounds, or even therapeutic antibodies, into the brain through the BBB. Some such receptors include LRP-1 and transferrin, which are found in this chip, as in real life. This could allow therapeutic antibodies targeting transferrin receptors to be shuttled across the BBB, without compromising its integrity.
Future research will aim at finding new targets for such drugs and molecules to be carried across the barrier, while also developing human antibodies to target known shuttle molecules that are more specific to the brain. The outcome, they hope, will be the ultimate development of shuttles “offering exceptional efficacy and engineering flexibility for incorporation into antibody and protein drugs, because this is so badly needed by patients and the whole field.”
Theranostic nanoparticles are another goal that researchers in this field are working towards to help treat the CNS cancer called medulloblastoma, as they could provide a new drug delivery platform for this difficult-to-treat tumor as well as a screening approach that is closer to actual physiological conditions.
The chip could also help understand neurodegenerative conditions and develop personalized medicine. By providing a good working model for drug-BBB interactions at molecular and cellular levels, this invention could help streamline the very expensive process of drug development for the CNS by increasing the ease of screening.
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