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Trespassing the Barrier of the Brain with UltrasoundFocused ultrasound is a powerful tool for overcoming the immense hurdles in drug delivery that are posed in brain therapeutics. The Blood-Brain Barrier Physiology: Structure and Function The brain is critical to the function of the whole body. Thus, any compromise of the brain by toxic molecules circulating in the blood could be fatal to the organ-ism. As a consequence, the brain has its own unique defense system to protect against toxic molecules. This defense system is mainly composed of the blood-brain barrier (BBB), which, as its name denotes, is a barrier or “filter” between the blood circulation in the brain and the brain tissue itself. In other words, the brain is protected from toxic molecules more stringently than is the rest of the body because of the BBB.

The BBB constitutes a collection of different cells (Figure 1) that contribute to this structural and functional barrier. Each of these cells has its own function of either transporting a molecule from the blood circulation into the brain tissue or parenchyma or allowing a molecule to diffuse due to its specific size, just as any other filter would do. For example, the most common feature of the BBB are the tight junctions, which are specialized proteins that connect the cells along the inner linings of the blood vessels (also known as “endothelial cells”), thus allow-ing only very small molecules (<400 Da in molecular weight or <1 nm in size) to

cross (Pardridge, 2015). The endo-thelial cells also have membranes that further filter molecules. The combination of the tight junctions and those membranes provide the filtering mechanism of the BBB. Other cells such as astrocytes and pericytes serve as mechanical ab-sorbers and thus provide a protec-tive mechanism of the neurons to any mechanical effect (Abbott et al., 2006).

The BBB and NeurotherapeuticsAccording to the National Center of Health Statistics (http://www.cdc.gov/nchs/fastats/deaths.htm), more than 5.4 million Americans

are currently diagnosed with Alzheimer’s disease, 1 million with Parkinson’s dis-ease, 350,000 with multiple sclerosis, 20,000 with amyotrophic lateral sclerosis (ALS), and 10,000 with brain cancer. Worldwide, these diseases account for more

Elisa E. Konofagou

Postal: Department of Biomedical

Engineering and Department of Radiology

Columbia University 1210 Amsterdam Avenue

ET 351, MC 8904 New York, New York 10027

USA

Email:ek2191@columbia.edu

©2017 Acoustical Society of America. All rights reserved. volume 13, issue 4 |

Figure 1. The blood-brain barrier (BBB) and its constituents. The lumen is from a capillary of the circulatory system and the surrounding cells con-stitute the BBB. Brain parenchyma represents the tissues of the brain.

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Overcoming the Brain Barrier

than 25 million patients. Although great progress has been made in recent years toward the understanding of neurode-generative diseases, few effective treatments, and no cures, are currently available.

Moreover, aging greatly increases the risk of neurodegen-erative disease, and the average age of Americans is steadily increasing. Today, over 35 million Americans are over the age of 65. Within the next 30 years, this number is likely to double, putting more and more people at increased risk of neurodegenerative disease. Alzheimer’s disease, which has emerged as one of the most common brain disorders, se-verely affects the memory center of the brain, with pathol-ogy gradually spreading to most brain areas as the disease progresses. This pathology is characterized partly by deposi-tion of protein deposits (amyloid plaques) in the brain tissue but also in the blood vessels themselves (Iadecola, 2004).

By acting as a permeability barrier, the BBB impedes entry from blood to the brain, thus rendering many potent, neuro-logically active substances and drugs ineffective simply because they cannot be delivered to where they are needed to treat dis-eases in the brain. As a result, traversing the BBB remains the rate-limiting factor in brain drug delivery development.

A variety of approaches have been, and are being, developed to overcome the BBB for selective therapeutic treatment of brain pathologies. Over the past decade, numerous small- and large-molecule products have been developed for treat-ment of neurodegenerative diseases with mixed success. When administered systemically in vivo (e.g., by injection into the bloodstream or intravenously), the BBB inhibits their delivery to the regions affected by those diseases.

Indeed, a review of the Comprehensive Medicinal Chemis-try database indicates that only 5% of the more than 7,000 small-molecule drugs can be used to treat the central ner-vous system (CNS; Pardridge, 2015). With these, only four CNS disorders can be treated: depression, schizophrenia, ep-ilepsy, and chronic pain (Ghose et al., 1999; Lipinski, 2000). Moreover, despite the availability of pharmacological agents that could, hypothetically, treat potentially devastating CNS disorders including brain tumors and age-related neurode-generative diseases, they remain undertreated mainly be-cause of the impermeability of the BBB.

To be successful, a drug delivery system that overcomes the BBB requires transient, localized, and noninvasive targeting of a specific tissue region. These factors are critical in devel-oping such a system because the drugs of choice are likely

to be very powerful and could damage healthy tissue, with significant consequences for the brain and the body.

To date, none of the techniques clinically used or currently under research address these issues within the scope of the treatment of neurodegenerative diseases. As a result, the present situation in neurotherapeutics enjoys few success-ful treatments for most CNS disorders. Several pharmaceu-tical companies use the technique known as “lipidization,” which is the addition of lipid groups to the polar ends of molecules to increase the permeability of the agent (Fischer et al., 1998). However, the effect is not localized because the permeability of the drug increases not only in the targeted region but also throughout the entire brain and body. There can thus be a limit to the amount absorbed before the side effects become deleterious (Fischer et al., 1998).

A second set of techniques under study is neurosurgically based drug delivery methods that involve the invasive im-plantation of drugs into a region with a needle (Blasberg et al., 1975). The drug spreads through diffusion and is often localized to the targeted region because diffusion does not allow molecules to travel far from their point of release. However, such invasive procedures traverse untargeted brain tissue to get to the target region, potentially causing unnecessary damage.

Other techniques utilize solvents mixed with drugs or adju-vants (pharmacological agents) attached to drugs to disrupt the BBB through dilation and contraction of the blood ves-sels (Pardridge, 2015). However, this disruption is not local-ized within the brain, and the solvents and adjuvants used are potentially toxic. Although this technique may consti-tute a delivery method specific to the brain, it requires spe-cial attention to each type of drug molecule and a specific transport system, resulting in a time-consuming and costly process while still not being completely localized to the tar-geted region. As a result, none of the brain drug delivery techniques are routinely used in the clinic and the state of the art in the treatment of brain diseases remains stagnant.

Focused Ultrasound with MicrobubblesBBB thus remains a formidable obstacle in treating CNS disorders and millions of patients are undertreated at best. However, another approach, focused ultrasound (FUS), pro-vides that unique combined advantage of extracorporeal ap-plication with the capability of focalization through the in-tact skull, thus offering an unprecedented highly localizable drug delivery system.

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Localization of brain drug deliv-ery is extremely important because opening the BBB across the entire organ may expose critical brain re-gions to a drug that may have delete-rious effects. In addition, most of the aforementioned brain diseases are concentrated in specific brain struc-tures such as the hippocampus or striatum. FUS, in combination with microbubbles, therefore constitutes the only truly transient, localized, and noninvasive technique for open-ing the BBB. Due to these unique advantages over other ex-istent techniques, FUS may facilitate the delivery of already developed pharmacological agents and could significantly impact how devastating CNS diseases are treated.

To provide for localized delivery, FUS employs curved trans-ducers that transmit acoustic waves that converge only at the geometric focus of the transducer (Figure 2). Most of the en-ergy delivered during this sonication induces mechanical ef-fects, thermal effects, or both. When this technology is used at high intensities, it is referred to as high-intensity focused ultrasound (HIFU).

Microbubbles are gas-filled or protein- or lipid-shelled for-mations that can be formed, activated, and injected intra-venously as contrast agents to enhance ultrasound imaging and especially the vasculature. The combination of FUS with microbubbles allows the separation of the mechanical effect of cavitation to occur at low-peak negative pressures without incurring thermal effects and thus leading to reversible and safe opening of the BBB (Konofagou, 2012).

Brain-Blood Barrier Opening Using Focused Ultrasound and MicrobubblesDue to the high spatial resolution of the FUS methodology, the beam can be focused in a specific region of the brain such as the hippocampus, a key short-term memory cen-ter, and thus a drug delivery target in Alzheimer’s disease or to the caudate putamen, an important region for motor control and thus relevant in Parkinson’s disease. Delivery of molecules up to 20 nm in size has also been demonstrated (Chen and Konofagou, 2014). Most important, no neuronal or cellular damage within the range of peak rarefactional pressures of 0.3 to 0.45 MPa has been reported, and the bar-rier has been shown to close within 4-48 hours under these

conditions, thereby preventing other (unwanted) molecules from crossing the BBB at the region of opening by FUS.

The feasibility of the BBB opening through the intact skull and skin and the successful imaging of the BBB opening in the area of the hippocampus at submillimeter imaging reso-lution have been shown in both wild-type and transgenic animals including models of glioblastoma, Alzheimer’s, and Parkinson’s disease. Indeed, there have been several reports over the past decade or two of using FUS and microbubbles to disrupt the BBB. This includes studies for neuroprotection and neurorestoration in Parkinson’s disease in rodents (Sa-miotaki, 2015), amyloid reduction in Alzheimer’s disease in rodents (Jordão et al., 2013; Leinenga et al., 2016), and treat-ment of glioblastoma in patients (Carpentier et al., 2016).

Mechanism of Blood-Brain Barrier OpeningThere are two physical mechanisms for opening the BBB with FUS. The first is to use the ultrasound beam at lower pressures to induce a stable oscillation of the microbubble, also known as “stable cavitation.” The second is to use high-er pressures to increase the magnitude of oscillation of the bubble to the point that it surpasses the inertia of the fluid and collapses on itself. This is called “inertial cavitation.” Both stable and inertial cavitation can be used to induce BBB opening. Stable cavitation has the safest profile and has been successfully monitored transcranially in real time in large animals, including through the human skull (Wu et al., 2014; Karakatsani et al., 2017; Figure 3).

Molecular Delivery Through the Opened BBBThe delivery of many large pharmacological agents across the BBB using FUS and microbubbles has been demon-

Figure 2. A: Focused ultrasound (FUS) system for opening the BBB in mice in vivo. B: Drug de-livery uptake (“brighter” regions) in the left hemisphere of a mouse brain after BBB opening with FUS. Inset: contralateral (right) hippocampus (no FUS) in the same mouse.

A) B)

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strated in previous studies that include imaging contrast agents (Hynynen et al., 2001; Choi et al., 2007; Samiotaki et al., 2016), antibodies (Kinoshita et al., 2006; Raymond et al., 2008), growth factor proteins (Baseri et al., 2012; Samio-taki et al., 2015; Figure 4), stem cells (Burgess et al., 2011), and gene delivery vectors (Wang et al., 2015, 2017; Figure 5). Molecular delivery studies (Choi et al., 2011; Chen and Konofagou, 2014) have indicated that the size of the BBB opening increases with the ultrasonic pressure, allowing larger molecules to diffuse of several orders of magnitude when the pressure is sufficiently high.

The permeability of the barrier increases with both the pres-sure and microbubble size (Vlachos et al., 2010), indicating that the BBB opening occurs at multiple sites within the capillary tree and that the BBB opening is larger with larger microbubbles, most likely due to the larger area of contact between the bubble and the capillary wall.

Safety and Reversibility of Blood-Brain Barrier OpeningThe safe operating parameters of ultrasound exposure of brain cells have been identified (Konofagou, 2012). In sum-mary, BBB opening starts occurring at 0.3 MPa rarefactional

pressure amplitude and beyond (Figure 5). At pressures un-der 0.6 MPa, no extravasation of red blood cells (RBCs) or neuronal damage was observed in the regions of the hippo-campus exhibiting the most pronounced BBB opening. Be-yond 0.6 MPa, RBC extravasation was detected, and beyond 0.9 MPa, neuronal damage was observed. These preliminary findings suggest that there is overlap between the feasibil-ity and safety windows within the pressure range of 0.3-0.6 MPa, meaning that the BBB can be opened throughout the entire hippocampus without endothelial or neuronal dam-age at those pressures (Baseri et al., 2010). FUS-induced BBB opening was reported to close within 24 hours under specific parameters in rabbits (Hynynen et al., 2001), mice (Samio-taki et al., 2016), and monkeys (Marquet et al., 2014). Be-havioral studies in mice that survived over six months when BBB opening was performed every week for six months showed no evidence of behavioral or motor control damage (Olumolade et al., 2016). Similarly, repeated sonications in Rhesus macaques demonstrated no changes in behavioral or physiological health of the animals after monitoring for two years (Downs et al., 2015). A recent report indicated that highly overlapping FUS exposure regions could increase in-flammation, including ischemia immediately after exposure

Overcoming the Brain Barrier

Figure 3. A: Large-animal FUS BBB opening system. DOF, degrees of freedom. B: BBB opening detection (blue) in the orthogonal MRI planes. C: Real-time cavitation monitoring. SCDh, harmonic stable cavitation dose; SCDu, ultraharmonic cavitation dose; ICD, inertial cavitation dose.

Figure 4. Neurotrophic protein delivery. A: Contrast-enhanced MRI of a Parkinsonian mouse model. B: Optical imaging showing protein uptake. C: Histological examination of protein uptake (brown tint) by neurons (arrows). D: Cell uptake in the striatum. RET, cell mem-brane receptor; ERK1/2, cytoplasm; CREB, neuronal nucleus. NTN, overall neurturin.

A)

B)

C)

A) B)

C) D)

Winter 2017 | Acoustics Today | 25

without investigating long-term effects (Kovacs et al., 2017). However, several groups have repeatedly shown that such an inflammation can be reversed after the opening depending on the pressure used (Sun et al., 2015).

Over the past couple of decades, therapeutic ultrasound has morphed into a formidable technology that has impacted several fields from dental care and alleviation from Parkin-sonian tremors to cancer therapies and treatment of kidney stones (see article by Simon, Maxwell, and Bailey in this is-sue of Acoustics Today). The brain, similar to the lungs, has always been an organ remotely related to ultrasound because of the limitations in its imaging. Not anymore. Because of the transcranial capabilities of ultrasound and the advent of MRI and CT imaging, therapeutic ultrasound on the brain has not only been shown feasible but has also been integrat-ed in the clinic for ablation of essential tremors.

However, as described herein, therapeutic ultrasound could also prove pivotal in brain drug delivery. FUS currently con-stitutes the sole technique that can noninvasively, safely, and transiently open the formidable frontier in brain drug deliv-ery, the BBB. It has even been shown that it can stimulate the immune system to facilitate toxic protein reduction that is associated with Alzheimer’s and to induce immunotherapy of cancer. More importantly, it constitutes a technology that can be combined with any drug already available to treat neurological disease. Therefore, given the immense need of

Figure 5. A: Gene delivery through the BBB (BBB-opened; left) vs. contralateral (unopened; right) brain hemispheres in two separate regions of the brain (top and bot-tom). Green, virus-transduced cells. AAV, adeno-associated virus. Gene-transduced neurons (B; green) and astrocytes (C; green; 100×) in the BBB-opened region from the left panel. Scale bars, 150 μm.

A) B)

C)

treating these currently intractable diseases over the next few years, ultrasound is expected to be readily available and in-valuable in the clinic for patients suffering from these condi-tions. It will signify the dawn of an exciting, new era in both acoustics and healthcare.

AcknowledgmentsThe work shown here was performed by Babak Baseri, Cher-ry Chen, Hong Chen, James Choi, Thomas Deffieux, Karen Duff, Vincent Ferrera, Fabrice Marquet, Gesthimani Samio-taki, Oluyemi Olumolade, Scott Small, Tobias Teichert, and Shih-Ying Wu. The research was supported by Grants R01-EB-009041 from the National Institute of Biomedical Imag-ing and Bioengineering, R01-AG-038961 from the National Institute on Aging, and R21-EY-018505 from the National Eye Institute, National Institutes of Health; National Science Foundation CAREER Award 0644713; the Kinetics Founda-tion; and the Kavli Institute.

Biosketch

Elisa Konofagou is the Robert and Mar-garet Hariri Professor of Biomedical En-gineering, professor of radiology, and director of the Ultrasound and Elastic-ity Imaging Laboratory, Columbia Uni-versity, New York. Professor Konofagou has coauthored over 170 peer-reviewed

journal articles and is a recipient of the National Science Foundation-CAREER Award, the National Institutes of Health Nagy Award, and awards from the American Heart Association, Acoustical Society of America (ASA), Ameri-can Institute of Ultrasound in Medicine, Wallace H. Coulter Foundation, Bodossaki Foundation, and Radiological Soci-ety of North America. Elisa is a fellow of the ASA and the American Institute of Biological and Medical Engineering.

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Overcoming the Brain Barrier