A prominent difference between physiology of mammals and other species is the presence of a cerebral vascular wall. The barrier, known as the Blood-Brain Barrier (BBB), is part of the brain’s natural defense. Although the BBB prevents harmful chemicals and viruses from entering the brain, it also prohibits many drugs from accessing the brain through vessels. As a result, doctors often need to inject drugs directly into the brain, which is an invasive approach that requires traumatic surgery on the skull. To circumvent this, scientists have developed a variety of ways to pass through the BBB in the recent years.
The Blood-Brain Barrier
The brain is protected by a layer of tightly joined endothelial cells that align on the blood vessels of the brain, which keeps harmful substances such as pathogens and toxins from entering the brain’s extracellular fluid. Although it allows passage of some compounds such as water, gases, hydrophobic molecules by passive diffusion, and glucose and amino acids by selective transport, it prevents the entry of a wide variety of microscopic objects, large or hydrophilic molecules, and certain toxins. Astrocytes are considered to be responsible for providing biochemical support to these endothelial cells and certain transmembrane proteins are responsible for stitching together the junctions between the cells. The main purpose of the BBB is to protect the brain from foreign substances as well as from hormones and neurotransmitters in the rest of the body, thereby maintaining a constant environment for the brain .
Targeted Delivery Via Microcapsules
The isolative function of the BBB often restricts flow of essential drugs to the brain. Therefore, to ensure that enough drugs have entered the problematic area of the brain, doctors often have to add a large quantity of drugs which consequently lead to different side-effects. Researchers recently found a more effective approach of delivering drugs via microcapsules. The microcapsules are made up of liquid crystal polymers (LCPs), which are a class of partially crystalline aromatic polyesters, substances known for their high mechanical strength, extreme chemical resistance, and inherent flame retardancy. These characteristics make LCPs suitable for making electrical and mechanical devices that require high strength and inertness. In this method, chemotherapy drugs are delivered using implantable microcapsules that are made of liquid crystal polymers. By using 1.5-milliliter capsules, the drugs diffuse through small holes in the BBB. Through this method, doctors can accurately control the release rate of the drug over long periods of time.
Compared to intravenous injection of excessive amounts of drugs, this method is more controllable and reduces side-effects to its accuracy. Michael Lim, an associate professor of neurosurgery at Johns Hopkins, believes that this approach has a significant influence on patients who have their brain metastases surgically taken out to kill remaining tumor cells via the implantation of microcapsules .
Nanoparticles and Ultrasound
Since crossing the Blood-Brain Barrier is difficult, how about opening it? This was the initial idea behind the FUS-BBB opening technique. By using focused ultrasound (FUS) exposure in the presence of gas-filled microbubbles, scientists are able to open the BBB temporarily. First, patients are injected with the microbubbles which will travel through the bloodstream to all parts of the body, including the blood vessels that wrap around the brain. Then, they will wear a cap that contains transducers that direct ultrasound waves, which will be concentrated inside the body. The concentrated applied ultrasound causes the bubbles to vibrate, expanding and contracting at a rate of about 200,000 times a second, which loosens the tight junctions of the cells comprising the blood-brain barrier. As a result, high concentrations of chemotherapeutics and even relatively large molecules can enter targeted tissues. The BBB begins to close immediately after the ultrasound is turned off, which means that there are very few side effects and almost no long-term effects on ongoing brain activity. Most importantly, the treatment is painless and non-invasive and is one of the most promising techniques of overcoming the BBB .
In fact, this technique has already been tested on a human patient as part of a pilot study. Neurosurgeon Todd Mainprize, MD., physicist Kullervo Hynynen, PhD., and their team were able to deliver the chemotherapy agent doxorubicin to the brain of a patient with a malignant brain tumor. Although this was a pioneer trial in humans, Dr. Hynynen had been performing similar preclinical studies which had shown that the combination of ultrasound waves and microbubbles might not only allow for more effective drug delivery but also stimulate the brain’s natural responses to fight disease .
Since it’s so hard for large particles to pass through the BBB, why not try using nano-sized viruses as a vector instead? The idea of transplanting targeted DNA into viruses has always been a hot topic in research. However, their lack of gene mutation stability and damage to the human body has always been a major concern. Adeno-associated viruses (AAV) are non-pathogenic without the presence of adeno-viruses, therefore they always cause no symptoms in human body. In addition, AAV is a special virus that has the ability to stably integrate its genetic material into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19. This factors indicate that AAV it is a very good media for transplanting DNA.
This June, a research paper published by neuroscientist Viviana Gradinaru and her colleagues at the California Institute of Technology showed that an AAV strain named AAV-PHP.B most reliably crossed the BBB. The team then encoded green fluorescent proteins in the aforementioned strain in order to evaluate the success rate by tracking the green glowing growth in neurons. This experiment shows impressive results as the green glowing effect can last for a year. In the future this method has large potential to help treat patients having problems with gene mutations, peripheral nervous system etc .
Liposomes are artificially-synthesized membranes composed of one or more lipid bilayers. Because they can carry hydrophilic molecules within their membrane and hydrophobic molecules within the lipid bilayer, they are a powerful tool for drug delivery to the nervous system. With different molecules in the mixture used to synthesize lipids, the properties of the liposomes can be easily manipulated to ensure optimal uptake of its contents by the brain. The use of surface molecules that will be detected by BBB receptors (including transferrin and insulin) is essential to ensure that the BBB will take in the contents of the liposomes. Researchers have found that liposomes lead to a tenfold increase in the uptake of certain drugs, while liposomes designed to express transferrin on their surface lead to a seventeen-fold increase.
One emerging technique to promote the uptake of drugs involves the creation of immunoliposomes, which have antibodies attached to their surface to improve targeting to the BBB. Much like their counterparts without antibodies attached, immunoliposomes show considerable promise. A notable study used immunoliposomes containing an expression plasmid for Tyrosine Hydroxylase, which would reverse a mutation that prevents the production of normal dopamine, was able to target the striatum of mouse models of Parkinson’s Disease and restore normal enzymatic function .
One emerging technique to allow the entry of drugs into the brain is the use of shuttle peptides. While the FUS-BBB opening technique involves increasing the permeability of the BBB to all compounds, even for a short period of time, it carries the risk of allowing the entry of cytotoxic particles. Thus, researchers have more recently began to focus on allowing the particles to readily diffuse directly through the endothelial membrane.
In 2007, a key paper found that RVG29 was capable of selectively transporting molecular cargoes into the brain. Many shuttle peptides with high specificity interact directly with transporters, however, others can bind to gangliosides (lipids with multiple sugars attached to the head group) to mediate the transport of nanoparticles across the cell membrane. With such techniques available, medications can be more readily transported across the BBB and lead to improved treatment outcomes.
According to the Royal Society for Chemistry, “Peptides are more affordable, easier to characterize and to link to nanocarriers or proteins.” In preclinical testing within the laboratory, an increase in therapeutic effect was seen after shuttle peptides were used in various animal disease models, ranging from brain tumor, epilepsy, lysosomal diseases to neurodegenerative disorders. Although these small biological delivery men have accomplished a great feat, more research still needs to be done in order to increase the efficiency and selectivity of these peptides .
The Significance of Crossing the BBB
Currently, an estimated 98 percent of potential drug treatments for brain disorders are unable to penetrate blood brain barrier.  Bypassing the BBB is crucial for these life-saving drugs to reach specific targets in the brain effectively and these non-invasive techniques are especially demanded because the constant delivery of a drug should not affect ongoing brain activity. For example, antibodies that could possibly wipe out the amyloid plaques in the brains of patients with Alzheimer’s diseases are not able to cross the BBB. A recent study at the Queensland Brain Institute in Australia showed that opening the BB with focused ultrasound reduced amyloid plaques and improved memory in a mouse model of Alzheimer’s, which may open doors for future clinical applications .
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Jacob Umans is an aspiring physician-scientist in the Stanford University Class of 2020. As a cofounder of the IYNA, he is passionate about science education and hopes to share his excitement about all subfields of neuroscience -- especially glial biology and neuroimmunity -- with students around the world. He hopes to go on to earn an MD/Ph.D. after graduating from Stanford and to use his clinical experience develop a research focused on developing a better understanding of and improved therapies for neurodegenerative diseases. Outside of neuroscience, Jacob is an avid fan of puns, table tennis, and reading.