aryal et al. - ultrasound-mediated blood-brain barrier disruption

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Advanced Drug Delivery Reviews xxx (2014) xxxxxx

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Contents lists available at ScienceDirect

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j ourna l homepage: www.eUN4.6.3. Treatment evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Going forward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .CO4.5. Delivery of imaging/therapeutic agents and tests in animal disease models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4.5.1. Delivery of therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.5.2. Disease models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4.6. Methods to plan, monitor, and evaluate FUS-induced BBB disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.6.1. Treatment planning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.6.2. Treatment monitoring and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 This review is part of the Advanced Drug DeliverUltrasound triggered drug delivery. Support: This work was supported by NIH grants P0P41RR019703, and R25CA089017.

Corresponding author at: 75 Francis Street, Boston, M278 0605; fax: +1 617 525 7450.

E-mail address: [email protected] (N. McDanno

0169-409X/$ see front matter 2014 Published by Elsehttp://dx.doi.org/10.1016/j.addr.2014.01.008

Please cite this article as: M. Aryal, et al., Ultsystem, Adv. Drug Deliv. Rev. (2014), http://Rother factors on BBB disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.2. Optimal parameters? . . . . . .4.3. Potential mechanisms . . . . . .1. Introduction . . . . . . . . . .2. Methods for drug delivery in the br

2.1. Invasive approaches to brain2.2. Transvascular brain drug th2.3. Transvascular brain drug th

3. Focused ultrasound . . . . . .4. Ultrasound-induced BBB disruption

4.1. Effect of ultrasound parameREC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0elivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0iopharmaceutical approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0BB disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Contents T

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Ultrasound-mediated bloodbrain barrier disruption for targeted drugdelivery in the central nervous system,

Muna Aryal a,b, Costas D. Arvanitis b, Phillip M. Alexander c, Nathan McDannold b,a Department of Physics, Boston College, Chestnut Hill, USAb Department of Radiology, Brigham & Women's Hospital, Harvard Medical School, Boston, USAc Department of Engineering Science, Brasenose College, University of Oxford, Oxford, UK

a b s t r a c ta r t i c l e i n f o

Article history:Accepted 14 January 2014Available online xxxx

Keywords:BrainDrug deliveryUltrasoundMicrobubbles

The physiology of the vasculature in the central nervous system (CNS), which includes the bloodbrain barrier(BBB) and other factors, complicates the delivery of most drugs to the brain. Different methods have beenused to bypass the BBB, but they have limitations such as being invasive, non-targeted or requiring the formula-tion of new drugs. Focused ultrasound (FUS), when combined with circulating microbubbles, is a noninvasivemethod to locally and transiently disrupt the BBB at discrete targets. This review provides insight on the currentstatus of this unique drug delivery technique, experience in preclinical models, and potential for clinical transla-tion. If translated to humans, this methodwould offer a exiblemeans to target therapeutics to desired points orvolumes in the brain, and enable the whole arsenal of drugs in the CNS that are currently prevented by the BBB.

2014 Published by Elsevier B.V.y Reviews theme issue on

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rasound-mediated bloodbradx.doi.org/10.1016/j.addr.201elivery Reviews

l sev ie r .com/ locate /addr481. Introduction

49Thebloodbrain barrier (BBB) is a specialized non-permeable barrier50in cerebral microvessels consisting of endothelial cells connected51together by tight junctions, a thick basement membrane, and astrocytic52endfeet. The tight junctions between the endothelial cells, together with

in barrier disruption for targeted drug delivery in the central nervous4.01.008

T53 an ensemble of enzymes, receptors, transporters, and efux pumps of54 the multidrug resistance (MDR) pathways, control and limit access of55 molecules in the vascular compartment to the brain by paracellular or56 transcellular pathways [1]. The BBB normally protects the brain from57 toxins, and helps maintain the delicate homeostasis of the neuronal58 microenvironment. However, it also excludes 98% of small-molecule59 drugs and approximately 100% of large-molecule neurotherapeutics60 from the brain parenchyma [2,3]. Only small-molecule drugs with high61 lipid solubility and a molecular mass under 400500 Da can cross the62 BBB in pharmacologically signicant amounts, resulting in effective63 treatments for only a few diseases such as depression, affective disor-64 ders, chronic pain, and epilepsy. Given the paucity of small-molecule65 drugs effective for CNS disorders, it is clear that the BBB is a primary66 limitation for the development and use of drugs in the brain. Overcoming67

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100implanting drug-exuding devices. With such techniques, therapeutic101benets have been shown for brain tumors and other disorders102[1417]. However, because of their invasiveness, there are some risks103of infection or brain trauma, and theymay not be amenable for repeated104treatments or for drug delivery to large areas of the brain. It can also be a105challenge to control the drug distribution, as drug concentrations106decrease exponentially from the injection or implantation site [18].107When convection-enhanced diffusion is used, the infused agents are108delivered preferentially along white matter tracts [19], which may not109be desirable.110Another approach for bypassing the BBB is to introduce drugs into111the cerebrospinal uid (CSF) via intrathecal or intraventricular routes.112It then follows the ow patterns of the CSF and enters the brain paren-113chyma via diffusion. This approach has been successful in cases where114

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this hindrance could mean potential therapies for a wide range ofdisorders, including Alzheimer's andHuntington's diseases, amyotrophiclateral sclerosis (ALS), neuro-AIDS, stroke, brain or spinal cord trauma,autism, lysosomal storage disorders, fragile X syndrome, inheritedataxias, and blindness.

Tumors, particularly those in the brain also face challenges foreffective drug delivery. While the blood vessels in most primary andmetastatic brain tumors are often somewhat permeable from the lackof a fully formed BBB, inltrating cancer cells at the tumor marginsand small metastatic seedsmay be protected by the BBB of surroundingnormal tissue [4]. Glioblastomas in particular are highly inltrative, andcommonly recur after localized treatments such as conformal radiother-apy or surgery. Relapse usually occurs within a few centimeters of thetreatment site [57]. Furthermore, their vascular permeability is hetero-geneous, and additional barriers to drug delivery include increasedinterstitial pressures [8] and drug efux pumps that contribute totheir multidrug resistance phenotype [9]. As for metastatic tumors,work in mice suggests that the bloodtumor barrier (BTB) is onlypartially compromised in breast adenocarcinoma brain metastases,and that toxic concentrations of chemotherapy are only achieved in asmall subset of tumors that are highly permeable [10]. Also, systemicdrug accumulation in brain metastases can be substantially less thanin extracranial metastases [10]. Thus, the BTB is a hindrance to effectivedrug delivery similarly to the BBB.

2. Methods for drug delivery in the brain

In order to overcome these limitations, it is necessary to either bypassthese vascular barriers altogether, or to facilitate passage across it viacontrolled exploitation of endogenous transport mechanisms. Differentmethods have been explored to bypass the BBB (or the BTB) (Table 1)[1113]. While these methods are promising, they also have limitations.

2.1. Invasive approaches to brain drug delivery

High local drug concentrations can be achieved by inserting a needleor catheter into the brain and directly injecting or infusing drugs or by

Table 1Different methods investigated to get around the BBB to deliver drugs to the brain.

Method Advantages

Direct injection, convection-enhanceddelivery, implantable devices

High local drug concentrations can be achieveadministration avoided.

Intrathecal, intraventricular injection Effectively delivers drugs to subarachnoid spacTrans-nasal delivery Noninvasive; easy to administer; repeatable.BBB disruption via arterial injection ofosmotic solution or other agents

Effectively delivers drugs to large brain regionexperience.

Modication of drugs to cross barrierthrough endogenous transportmechanisms

Easily administered; delivered to whole brain.

BBB disruption via FUS andmicrobubbles

Noninvasive; readily repeatable; can target druvolumes; can control magnitude of disruptiondrug-loadedmicrobubbles ormagnetic particlePlease cite this article as: M. Aryal, et al., Ultrasound-mediated bloodbrasystem, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/j.addr.201ED P

ROOFthe target is in the subarachnoid space [20], but drug diffusion dropsoff exponentially from the brain surface and penetration into the brain

parenchyma can be limited [11]. It is also possible to deliver drugstransnasally from the submucus space into the olfactory CSF [2124].This approach has advantages of being noninvasive and being relativelyeasy to administer. However, only small drug volumes can be deliveredand interindividual variability and other factors may pose challenges tothis procedure [24]. Nevertheless, the technique is a promising route tobypass the BBB and is currently being investigated by numerousresearchers.

2.2. Transvascular brain drug therapy: Biopharmaceutical approaches

Anumber of approaches have been investigated to develop ormodifydrugs that can cross the BBB. While these methods are highly promisingand offer the ability to easily administer drugs to the CNS as in otherorgans, they do require the expense and time of developing new agents,and they result in drugs being delivered to the entire brain, which maynot always be desirable.

Converting water-soluble molecules that would not ordinarily crossthe BBB into lipid-soluble ones is one approach to brain drug therapy.This can be achieved by the addition of lipid groups, or functional groupssuch as acetate to block hydrogen bonding. The molecule would thenundergo passive diffusion across the BBB. An example of this is the con-version of morphine to heroine by the acetylation of two hydroxylgroups, which results in the removal of the molecule from hydrogenbonding with its aqueous environment [25]. Although utilized by thepharmaceutical industry, this approach has limited applicability todrugs greater than 400450 Da [12,26].

Another approach involves utilizing the large variety of solute carrierproteins (SLC) on the endothelial surface that specically transportmany essential polar and charged nutrients such as glucose, aminoacids, vitamins, small peptides, and hormones transcellularly acrossthe BBB [27]. These transporters move the solute into the cytoplasmwhere they await another SLC at the opposite cell membrane to exocy-tose them into the brain parenchyma. An example of SLC used for braindrug therapy is the large neutral amino acid transporter type 1 (LAT1),

Disadvantages

stemic Invasive; side effects; challenging to control; not readilyrepeatable.

rain surface. Little drug penetration beyond brain surface; invasive.Small volume of drug delivered; interindividual variability.

rge clinical Invasive; requires general anesthesia; side effects; not readilyrepeatable.Requires systemic administration; expensive; each drug requiresnew development; clinical data lacking.

livery to desiredn be combined withadditional targeting.

Requires systemic administration; currently technicallychallenging; large volume/whole brain disruption unproven; noclinical data.in barrier disruption for targeted drug delivery in the central nervous4.01.008

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which transports the amino acid Parkinson's drug L-dopa across theBBB. Once across, it is converted to dopamine by aromatic amino aciddecarboxylase, and can then bind to its target receptor. Dopaminebeing water-soluble cannot cross the BBB [26,28].

Finally, the molecular targeting of endothelial-surface receptors,colloquially termed the TrojanHorse approach, is yet another paradigmin drug transport across the BBB. This involves using a targeting ligandsuch as a serum protein, monoclonal antibody, or other high afnitytargeting molecule that binds to its receptor and activates endocytosisof the complex into a vesicle that is transported across to, and releasedfrom the opposite pole (i.e., transcytosis). In theory, if the ligand is chem-ically linked to a drug or drug carrier, it too is transported across the BBB.Over the last twodecades, a number of animal studies have suggested thetransport of antineoplastic drugs, fusion proteins, genetic therapies (plas-mid vectors, siRNA), liposomes, and nanoparticles by this mechanism[2932]. For transcytosis to occur, it requires that the endosome notfuse with lysosomes while in the cytoplasm, which would degrade theinternalized macromolecules. Unlike other tissues, endothelial cells inbrain capillaries appear to have low levels of endosome fusion withlysosomes, facilitating transport of necessary substances through thetranscellular route [3335].

2.3. Transvascular brain drug therapy: BBB disruption

Others have investigatedmethods to temporarily disrupt the BBB toenable CNS delivery of circulating agents. One such technique investi-gated intensively for several decades is the intraarterial injection ofhyperosmotic solutions such asmannitol. This procedure causes shrink-age of endothelial cells and consequent stretching of tight junctions[3639] through which drugs may pass. This method has been shownrepeatedly to enhance delivery of therapeutic agents to brain tumors,and several promising clinical trials have been performed [4045].Other agents such as bradykinin have also been investigated [4649].While such methods can be an effective means to deliver drugs tolarge brain regions, they are invasive procedures that require generalanesthesia, and can have side effects. For example, one study reportedfocal seizures in 5% of patients who received osmotic BBB disruption[40], and others have noted vasovagal response with bradycardia andhypotension [39]. Having a less-invasive way to achieve this disruptionwould be desirable.

The use of ultrasound,when combinedwith circulatingmicrobubbles,offers a potential way to disrupt the BBB in a targeted, noninvasive, andrepeatable manner to deliver a wide range of drugs to the brain and tobrain tumors. Below, we review the literature on this technique,(i) describing how it is performed, (ii) how different parameters effectthe BBB disruption, (iii) what has been delivered in preclinical studies,and (iv) methods that can be used to guide the procedure. While todate the technique has only been performed in animals, it is clear thatit holds great promise for the treatment of a wide range of CNS disor-ders. If successfully translated to the clinic, it offers a means to targetdrugs, biomolecular therapies, and perhaps cellular therapies to desiredbrain regions while sparing the rest of the brain from unnecessaryuptake. The technique also offers the potential to control the magni-tude of BBB disruption at each focal target through modicationof the ultrasound parameters, enabling a level of control over drugdelivery that is not available with other technologies. This exibility,along with its noninvasiveness, lack of need for general anesthesia,and amenability to be readily repeatedmake FUS a potentially transfor-mative technology.

3. Focused ultrasound

An ultrasound eld can be noninvasively focused deep into the bodyand used to induce a broad range of bioeffects through thermal ormechanical mechanisms. FUS has been investigated since the 1940's

for noninvasive ablation in thebrain, as a potential alternative to surgical

Please cite this article as: M. Aryal, et al., Ultrasound-mediated bloodbrasystem, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/j.addr.201ED P

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resection and radiosurgery [5053]. Until recently, clinical testingrequired a craniotomy to allow for ultrasound propagation into thebrain [54,55] because of ultrasonic heating of the skull, and beam aberra-tion caused by the skull's irregular shape and large acoustic impedance.

In the past decade, FUS thermal ablation systems have been devel-oped that overcome these obstacles produced by the skull [56]. Theyreduce skull heating through active cooling of the scalp and a transducerdesign with a large aperture to distribute the ultrasound energy over alarge skull region, and they correct for beam aberrations using a phasedarray transducer design.When combinedwithmethods that use acousticsimulation based on CT scans of the skull bone to determine the phaseand amplitude corrections for the phased array [57,58] andMR temper-ature imaging (MRTI) to monitor the heating [59], a completely nonin-vasive alternative to surgical resection in the brain becomes possible.These systems use very high intensities to enable thermal ablationthrough the human skull, and are currently in initial human trials[6063].

The effects of FUS can be enhanced by combining the ultrasoundexposures (sonications) with preformed microbubbles that are com-mercially available as ultrasound imaging contrast agents. They consistof semi-rigid lipid or albumin shells that encapsulate a gas (typicallya peruorocarbon), range in size from about 110 m, and areconstrained to the vasculature. The microbubbles concentrate theultrasound effects to the microvasculature, greatly reducing the FUSexposure levels needed to produce bioeffects. Thus, with microbubblesone can apply FUS transcranially without signicant skull heating.

When microbubbles interact with an ultrasound eld, a range ofbiological effects have been observed [64]. Depending on their size,the bubbles can oscillate within the ultrasound eld, and they cangrow in size via rectied diffusion. They can interact with the vesselwall through oscillatory and radiation forces [65,66]. They also canexert indirect shear forces induced by micro-streaming in the uidthat surrounds them [67]. At higher acoustic pressures, they cancollapse during the positive pressure cycle, a phenomenon known asinertial cavitation, producing shock waves and high-velocity jets [65],free radicals [68], and high local temperatures [69,70]. Themicrobubblesused in ultrasound contrast agents can presumably exhibit these behav-iors, either with their shells intact or after being broken apart by theultrasound beam and their gas contents released.

4. Ultrasound-induced BBB disruption

Since the early years of investigation into ultrasound bioeffects onthe brain, several studies have noted localized BBB disruption, eitheraccompanied with tissue necrosis or without evident tissue damage[52,7176]. None of these early studies however, elucidated sonicationparameters that could repeatedly and reliably produce BBB disruptionwithout occasionally producing lesions or necrosis.

In 2000 our laboratory found that if short ultrasound bursts are pre-ceded by an intravenous injection of microbubble contrast agent, theBBB can be consistently opened without the production of lesions orapparent neuronal damage [77]. The circulating microbubbles appearto concentrate the ultrasound effects to the blood vessel walls, causingBBB disruption through widening of tight junctions and activation oftranscellular mechanisms, with little effect on the surrounding paren-chyma [78]. Furthermore, the opening occurs at acoustic power levelorders of magnitude lower than was previously used, making thismethod substantially easier to apply through the intact skull. For BBBdisruption, the sonications have been typically applied as short (~120 ms) bursts applied at a low duty cycle (15%) for 0.51 min. Witha few simple modications to enable low-intensity bursts, existingclinical brain FUS systems can be used for BBB disruption [79]. Clinicaltranslation may also be possible using simpler FUS systems [80].

Fig. 1 shows examples of targeted BBB disruption in amacaque fromour institution using a clinical transcranial MRI-guided FUS system274(ExAblate, InSightec, Haifa, Israel) [79]. The device uses a hemispherical


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1024-element phased array operating at 220 kHz, and is integratedwith a 3T MRI scanner. The focal region can be electronically steeredto different locations using this array without physically moving thetransducer. Volumes can be targeted by systematically steering thefocal point to different targets, enabling one to deliver drugs to desiredbrain regions. Fig. 2 shows an example of such volumetric FUS-induced BBB disruption. BBB disruption was evaluated using two MRIcontrast agents and with the vital dye trypan blue. Note the lack of con-trast enhancement in white matter despite evident staining with thedye. This difference is presumably due to the lower vascular density inwhite matter compared to gray matter.

4.1. Effect of ultrasound parameters and other factors on BBB disruption

A number of sonication parameters can be varied in ultrasonic BBBdisruption. Each parameter variationmay impact the threshold pressureamplitude needed to disrupt the BBB, the magnitude of its disruption,and the resultant drug quantity delivered to the brain parenchyma. Asdetermined from a number of studies, parameter variations and theireffects are listed in Table 2. These studies used an MRI contrast agent,uorescent probe, or drug to evaluate the BBB disruption. Given thelarge parameter space, and different techniques and criteria used toevaluate the disruption (each with different sensitivities), it can bechallenging to compare results from different laboratories. Such com-

Fig. 1. Contrast-enhanced T1-weighted MRI of the brain of a rhesus macaque showing enhAxial; middle: Sagittal; right: Coronal). Enhancement of this MRI contrast agent, which nowere sonicated using a 220 kHz clinical MRI-guided FUS system along with an infusion owide and4 mm long. Note the leakage of contrast agent into the cingulate sulcus evident inmapping during microbubble-enhanced FUS [176] (bar: 1 cm).UNCOparisons are additionally confounded by uncertain accuracies in esti-mates of acoustic pressure amplitude when sonicating through the

skull [81]. However, general trends can be observed.For a xed set of parameters, as one increases the pressure ampli-

tude, the magnitude of the BBB disruption increases, and at some levelit appears to saturate [8284]. Below some value, no disruption isdetected, and at some higher pressure threshold, vascular damage isproduced along with the disruption (see below). Such studies repeatedwhile varying a different parameter have shown that the threshold forBBB disruption depends strongly on the ultrasound frequency [85] andburst length [86]. Most experiments have been done with commercially-available ultrasound contrast agents that consist of microbubbles witha wide range of diameters. Experiments with microbubbles withnarrow size distributions suggest that the BBB disruption thresholdcan also be reduced by using larger microbubbles [8789].

By xing the pressure amplitude and varying each parameter, onecan evaluate their effects on the magnitude of the disruption. The mag-nitude has been found to increasewith the burst length up to a durationof approximately 10 ms, with further increases in burst length havinglittle or no effect [77,81,86,90,9092]. Several groups have shown thatthe disruption magnitude may be increased by using a larger dose of


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ultrasound contrast agent [83,9395] (although other works haveshown little or no effect [86,90]) or by using larger microbubbles[8789]. Pulse repetition frequency can also inuence the magnitudeof disruption up to a point [90,91], but other studies have seen nodependence [86]. Finally, the magnitude of the disruption can beincreased by increasing the sonication duration [84] or by repeating thesonication after some delay [96,97], but excessive durations may resultin tissue damage [84,97]. Factors such as using an infusion instead of abolus injection of microbubbles [98] and choice of anesthesia protocol[99] may also inuence the resulting disruption. Other factors such asthe delay between the microbubble injection and the start of sonication,andwhether the drug or tracer is administered before or after the sonica-tion may also be expected to have an effect. Additive effects have beenobserved when FUS-induced BBB disruption is combined with agentsthat affect vascular permeability [100102].

These trends observed in parametric studies are difcult to interpretwith condence since the exact mechanism by which microbubble-enhanced FUS induces BBB disruption is currently unknown (seebelow). They are perhaps consistent with the following notions. First,for BBB disruption to occur, the microbubbles oscillations may need toreach a certain minimal radius, which can be achieved by increasingthe pressure amplitude or by using larger microbubbles, and assumingthe bubbles grow during each burst via rectied diffusion, by decreasingthe ultrasound frequency or increasing the burst length. Next, in addi-

ement at four focal targets in the cingulate cortex after injection of Gd-DTPA. (Left image:ally does not extravasate into the brain, indicates areas with BBB disruption. Four targetsenity microbubbles. The dimensions of the disrupted spots were approximately 3 mmagittal image (arrow). These imageswere obtained in a study evaluating passive cavitation342tion to depending on the bubble size during its oscillation, the magni-343tude of the disruption depends on the number of sites on which the344microbubbles interact with the vasculature. The number of these sites345can be increased by increasing the microbubble dose, or by increasing346the sonication duration and/or number of bursts. Data showing a strong347dependence on burst length may also suggest that the threshold and348magnitude of the disruption depend on the amount of time the349microbubbles interact with the blood vessels during each burst. Pulse350repetition frequency may have an inuence if the microbubbles are351being fragmented or destroyed time may be needed to replenish352them if that is the case [103]. Finally, it appears that the magnitude of353the disruption can saturate at some level, and increasing the different354parameters has no additional effect.

3554.2. Optimal parameters?

356Overall, these studies have made it clear that BBB disruption is357possible over a wide range of exposure parameters. Disruption has358been demonstrated at frequencies between 28 kHz [104] and 8 MHz359[92], burst lengths as low as a few ultrasound cycles [90,91,98] up360to 100 ms [77], and over a range of pulse repetition frequencies,


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364maximize the window in acoustic pressure amplitude where robust365BBB disruption is possible without producing vascular damage. It will366be challenging to precisely estimate the pressure amplitude in the367human brain after transcranial sonication, and having the widest safety368margin possible will be desirable for clinical translation. How close the369FUS frequency is to the resonant size of the microbubbles may have370an impact on the width of this safety window. Additional important371criteria would be to optimize the frequency and transducer geometry372to produce the desired focal spot size, to effectively focus through the373skull with minimal distortion, and if a phased array transducer is used,374to be able to steer the focal region throughout the brain. It may also be375desirable to nd parameters that enable BBB disruption in the shortest376possible sonication time so that multiple targets can be targeted in a377

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microbubble doses, and sonication times. It is not clear what the opti-mal parameters are, or what criteria to use to establish them. In ourview, the primary consideration could be to nd parameters that

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411biologically-signicant shear stresses on the neighboring endothelium,412

Fig. 2. Demonstration of FUS-induced BBB disruption using contrast enhanced MRI andtrypan blue. (AC) Contrast-enhanced T1-weighted MRI after BBB disruption at sixvolumes in the cingulate cortex. At each volume, the focal regionwas steered electronicallyin sequence to nine targets in a 3 3 grid using a phased array. (A) Low-level enhance-ment observed with gadofosveset trisodium, an MR contrast agent that binds to albuminin the blood (MW of albumin: ~67 kDa); it was administered before sonication.(B) Enhancement after injection of Gd-DTPA (MW: 938 Da). The inset in (B) shows thesame view in T2-weighted imaging. The enhancement patterns correspond to regions ofcortical gray matter visible in T2-weighted imaging. (C) Sagittal view of Gd-DTPAenhancement, which included leakage of agent into a sulcus (arrow). (DE) VolumetricBBB disruption at three targets centered on the boundary between the cingulate cortexand white matter; from another experimental session in this animal. (D) T1-weightedMRI showing Gd-DTPA extravasation in the cingulate cortex, but not in the white matter.(E) Photograph of formalin-xed brain showing trypan blue extravasation into both thecingulate cortex and white matter. This differential enhancement between gray andwhite matter presumably reects differences in vascular density. The white matter com-ponent of two of these targets is shownwith increased image contrast in the inset to bettervisualize low-level trypan blue extravasation. (Scale bars: 1 cm). Reprinted from CancerResearch 2012; 72:36523663; 2012 American Association for Cancer Research. (Forinterpretation of the references to color in this gure legend, the reader is referred to theweb version of this article.)

Table 2Reported effects of different parameters on BBB disruption via FUS and microbubbles.

Parameter Effect on BBB disruption

Pressure amplitude Increase in BBB disruption magnitude as pressure amppressure amplitudes.

Ultrasound frequency Decrease in BBB disruption threshold as frequency deBurst length For burst lengths less than 10 ms, BBB disruption thre

[86,9092]; little or no increase in disruption magnituPulse repetition frequency BBB disruptionmagnitude increases as repetition frequ

magnitude [86].Ultrasound contrast agent dose Magnitude of BBB disruption increases with dose [83,Sonication duration Longer durations [84] or repeated sonication [96,97] inMicrobubble diameter Threshold for BBB disruption lower for larger microbuUltrasound contrast agent Similar outcomes reported forOptison andDenitym

Please cite this article as: M. Aryal, et al., Ultrasound-mediated bloodbrasystem, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/j.addr.201and the oscillations produce inward forces that in extreme cases can

litude increases; saturation at some point [8284]; vascular damage produced at high

creases; some evidence of improved safety for lower frequencies [85].shold increases and BBB disruption magnitude decreases as burst length is reducedde for longer bursts [77,81,90].ency increases up to a point [90]. Otherworks have observed no effect onBBB disruption

90,94,188]; other experiments have reported no effect [86].creasemagnitude of BBB disruption; damage reportedwith excessive sonication [84,97].bbles; disruption magnitude increased with larger microbubbles [8789].icrobubbles [189]. Sonovuemicrobubbles and research agents are also commonly used.ED P

ROOFreasonable amount of time, and tomaintain a safe dose ofmicrobubbles.

4.3. Potential mechanisms

Even though FUS exposures combinedwithmicrobubbles have beeninvestigated to disrupt the BBB in numerous studies, the exact mecha-nism to open BBB still remains unknown. It does appear that twoknown effects that can be induced by FUS, bulk heating and inertialcavitation, are not responsible. Initial studies on the method utilizedMRI-based temperature imaging [77] during the sonications, and nomeasureable heating was observed. Studies that recorded the acousticemissions during the sonications [105107] have found that BBBdisruption can be achieved without wideband acoustic emission,which is a signature for inertial cavitation [53]. It also may not be thesame mechanism utilized for so-called sonoporation, where transientpores in cell membranes created by sonication with microbubblesenable drugs to enter [108]. Those pores are rapidly resolved, whileFUS-induced BBB disruption lasts for several hours.

Fundamentally, we do not know if the FUS/microbubble interactionsphysically modify the vessel walls, or if they are triggering a physiolog-ical response that includes temporary BBB breakdown. As describedbelow, electron microscopy studies have shown delivery of tracersthrough widened tight junctions [78,109], which could be consistentwith a direct physical force pulling themapart, aswell as active transport[78,110]. Other work has shown the sonications can induce vascularspasm [111,112]. While the role of this spasm is not clear, it does makeclear that the sonications can trigger a physiological response.

In the absence of bulk heating and inertial cavitation,we are leftwithmechanical effects induced during the microbubble oscillations in theultrasound eld. A number of effects are produced with potential toinduce the observed BBB disruption. Microbubbles tend to move inthe direction of the wave propagation via acoustic radiation force [66],which will bring them in contact with vessel endothelium. Duringoscillation, the shell of the microbubble can break, the bubbles can befragmented into smaller bubbles, and they can grow via rectieddiffusion. Microstreaming due to microbubble oscillations can inducein barrier disruption for targeted drug delivery in the central nervous4.01.008

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413 pull the vessel wall inward [113]. Clearly, the behavior of a microbubble414 in an acoustic eld is complex, and it can be different in free uid than415 when constrained within a capillary [114].

416 4.4. Bioeffects induced by FUS and microbubbles

417 The BBB disruption can occur almost immediately with sonication418 [112] and appears to decay exponentially over several hours thereafter419 [77,82,96,115]. The amount of agent delivered across the barrier420 appears to bemuch larger in graymatter than in whitematter, presum-421 ably due to differences in vascular density [79]. Several studies have422 found that the barrier appears to be largely restored in approximately423 46 h [77,82,96,109,115,116]; other experiments have observed low-424 level disruption at 24 h after sonication or longer [89]. The source of425 this discrepancy is not clear, but it could be simply that more sensitive426 detection methods such as high-eld MRI combined with large doses427 of MRI contrast agent are capable of detecting low-level disruption428 missed in other works. The duration of the opening to different tracers429 appears to be reduced for larger tracers [115].430 This window in timewhere the barrier is open is thought to be good431 for the prospect of delivering even long-circulating drugs, but not so432 long as to produce concern of toxicity arising from chronic BBB break-433 down. Indeed, the appearance of the brain after BBB disruption in light434 microscopy appears to be normal [117], even after repeated weekly435 sessions [79]. Example histology obtained after BBB disruption is436 shown in Fig. 3. The only major feature that has been observed in437 many studies is the presence of tiny clusters of extravasated red blood438 cells (petechiae) [118,119]. It is thought that these petechiae are formed439 during inertial cavitation, andexperimentswherenowidebandemissions440 (a signature for inertial cavitation)were observed, no such extravasations441

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446failed to observe anythingmore thana few individual damagedneurons,447and long-term effects have not found evidence of neuronal damagewith448such sonications [118,120]. At excessive exposure levels, more severe449vascular damage, parenchymal damage, and neuronal loss can occur450[77,121].451Transmission electron microscopy (TEM) investigations have452demonstrated an increase of cytoplasmic vesicles in endothelium and453pericytes (suggestive of transcytosis), formation of trans-endothelial454fenestrae, widened tight junctions, and transport of serum components455across the BBB [78]. The use of a 44 kDa tracer molecule helped eluci-456date arterioles as the major sight of trans-endothelial vesicle transport457(followed by capillaries then venules), and showed extensive tracer458deposition in the endothelial paracellular space, basement membrane,459and surrounding brain parenchyma [110]. Finally, using immunogold460labeling, the disappearance of tight junction (TJ) proteins occludin,461claudin-5, and ZO-1 were shown, along with opened endothelial junc-462tions and tracer leakage at 14 h post-sonication [109]. The TJ proteins463reappeared at 6 and 24 h. Examples showing tracer penetration across464the BBB through widened tight junctions and vesicular transport are465shown in Fig. 4. Other work has shown down-regulation of the same466TJ proteins along with their mRNA, and recovery to normal levels at46712 h post-sonication [122]. Reorganization of connexin gap junction468proteins have also been reported [123]. An increase of endothelial469vesicles in normal [124] and tumor microvessels [125] have also been470observed on TEM with an up-regulation of caveolin proteins/mRNA,471suggesting that caveolae-mediated transcytosis (CMT) as a contributing472mechanism for permeability. These researchers also found increased473phosphorylation of Src and caveolin-1/2, noting that Src-induced phos-474phorylation of caveolins is a trigger for CMT [126].475Intracellular signaling cascades in response to mechanical stimula-476

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were observed [105]. Some have suggested that wideband emissionscan be observed without producing such petechiae [106]. While thepresence of these petechiae is undesirable, their impact on the brainmay be minimal. Investigations looking for apoptosis or ischemia,which may be expected if serious vascular damage were occurring,

Fig. 3. Normal-appearing histology after FUS-induced BBB disruption. This example was ogeniculate nucleus (LGN) in macaque. This target was sonicated approximately 2 h becontrast-enhanced T1-weighted MRI showing BBB disruption induced by sonicating ninhippocampus/LGN. (CD) High-magnication views showing normal-appearing layers in(E) and the pyramid layers (FG). (H) Blood vessels in the LGN with a few extravasated reextravasations were found. (BG: Nissl; H: H&E; scale bars: A: 1 cm, BD: 1 mm, EH: 20

for Cancer Research.

Please cite this article as: M. Aryal, et al., Ultrasound-mediated bloodbrasystem, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/j.addr.201Etion by FUS-induced BBB disruption is likely, but has only recentlybeen addressed. Increased phosphorylation of Akt and its downstreammolecule GSK3 has been shown in neurons anking the BBB disrup-tion at 24 h, well after tight junction reassembly [127]. Akt phosphory-lation has been implicated in neuroprotection after stroke [128], while

ned after volumetric sonication to induce BBB disruption in the hippocampus and lateralthe animal was sacriced and in seven prior sessions over several months. (A) Axial

rgets in a 3 3 grid. (B) Low-magnication microphotograph showing histology in theLGN. (EG) High-magnication views of hippocampus showing the granular cell layer

lood cells, presumably from the last sonication session. Only a very small number of such). Reprinted from Cancer Research 2012; 72:36523663; 2012 American Associationin barrier disruption for targeted drug delivery in the central nervous4.01.008

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7M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014) xxxxxxFig. 4.BBB permeability for horseradish peroxidase (HRP). (A) Photomicrograph showing pNo HRP passage to the basement membrane (arrowheads) or the neuropil [NP] can be see(B) A portion of a microvessel with adjacent nerve tissue from a sample obtained 1 h afterarrowheads. The tracer has inltrated the basement membrane [B] and the interstitial spobtained 2 h after sonication. The tracer is present in the junctional cleft (arrows), the ba(D) Full restoration of the tight junctional barrier function 4 h after sonication. Immersiothe tracer (black) lling the lumen [L] and being stopped at the rst tight-junction (arrow).membrane [B] or neuropil [NP]. E: endothelial cell cytoplasm; RBC: red blood cell in the lum2008; Copyright 2008World Federation for Ultrasound inMedicine & Biology. (E) Electrodemonstrated by the increased number of HRP-positive caveolae (arrows). Signicantly feModied from Ultrasound in Med. & Biol., Vol. 32, No. 9, pp. 13991409, 2006; Copyright UNCORRECTactivation of the p38 JNK MAP kinases promotes neuronal apoptosis

[129,130]. Alonso et al. showed increased protein ubiquitination inneurons not glia post sonication, no increase in heat shock proteins,and limited neuronal apoptosis at 24 h in areas staining positive forextravasated albumin [131]. Ca2+ signaling has also been suggested asbeing stimulated by FUS-induced BBB disruption. Specically, tempo-rary disruption of the endothelial plasmamembrane (i.e., sonoporation)can induce immediate transient changes of intracellular Ca2+ concen-tration in cells with direct contact with microbubbles, and delayed uc-tuations in nearby cells [132]. When factoring in uid shear induced inan in vitro ow channel (intended tomimic cerebral vessels), themem-brane disruption and Ca2+ transients were much lower [133].

Fig. 5. Vascular effects observed in real time during FUS-induced BBB disruption using in vivobefore, during, and approximately 20 min after sonication. Arterioles and veins (determined0.1 mL (2 mg/mL) 10 kDa, dextran-conjugatedAlexa Fluor 488 intravenously ~5 mins before imwas initiated and a 0.1 mL bolus (10 mg/mL) of 70 kDa, dextran-conjugated Texas Redwas deliof the eld occurred 12 s after the initiation of US (arrow). Beginning at 60 s and by 305 s, leavessel. (For interpretation of the references to color in this gure legend, the reader is referredModied from Journal of Cerebral Blood Flow & Metabolism 2007;27(2):393403; Copyright


RO

f a cross-sectionedmicrovessel and the surrounding nerve tissue from a nonsonicated area.e lumen [L] appears empty because the tracer was washed out during perfusion xation.ication. Passage of HRP (black color) through several interendothelial clefts is indicated by(arrows) in the neuropil. (C) A portion of longitudinally sectioned capillary in a sampleent membrane [B] and the interstitial spaces (asterisks) between myelinated axons [Ax].ation (instead of perfusion xation) was used in this brain and permits visualization ofenetration can be seen in the rest of the junctional cleft (arrowheads), nor in the basementcale bars: 200 nm.Modied fromUltrasound inMed. & Biol., Vol. 34, No. 7, pp. 10931104,icrograph of an arteriole 1 h after sonication at 0.26 MHz. An intense vesicular transport isvesicles were observed in capillaries and venules. L: lumen.06 World Federation for Ultrasound in Medicine & Biology.493Multiphoton microscopy (MPM) has provided useful insights into494the bioeffects of FUS-induced BBB disruption. Initial work with this495technique demonstrated arteriolar vasospasm in 14/16 mice lasting up496to 5 min (Fig. 5), and interrupted cerebral blood ow [111]. Although497this could cause ischemic injury, it has been noted that mice have498enhanced vasomotor excitability over other rodents, such as rats [134].499Indeed, a similar study in rats showed vasospasm in only 25% of the500vessels examined [112]. Initial work has also noted two forms of vessel501dye leakage, rapid focal microdisruptions (39 s) that were prevalent502at vessel bifurcations and slow disruptions that were observed as a503gradual increase in extravascular signal intensity [111]. Subsequent504work noted three rather than two leakage types: (1) fast, characterized

multiphoton microscopy. Each frame is a 615 615 m image acquired using a mouseby dye transit) are marked a and v respectively, in the rst frame. The animal receivedaging (green in images). Immediately after therst framewas taken, a 45-secUSexposurevered intravenously (red in images). Almost total occlusion of the large vessel in the centerkage in the green channel is apparent in the lower left of the eld, and around the centralto the web version of this article.)2006 ISCBFM.


TF

505 by rapid increase to peak intensity and rapid decrease, (2) sustained,506 described as rapid increase to peak which persisted for up to an hour,507 and (3) slow, a gradual increase to peak intensity [112]. The authors508 noted that differing vessel calibers have preferences for different leakage509 types, and interestingly, that distinct peak negative pressures also show510 preference for leakage types. Continuing work suggested correlation511 between fast leakage, common with high pressure amplitudes, and512 detachment of astrocyte endfeet from the vessel walls [135].

513 4.5. Delivery of imaging/therapeutic agents and tests in animal disease514 models

515 One advantage of this method for targeted drug delivery in the brain516 is that it appears to be drug neutral that is, it appears that many517 agents with a wide range of properties can be successfully delivered518 across the BBB and/or the BTB. A large number of imaging tracers519 (Table 3) and therapeutic agents (Table 4) which normally do not520

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549works have shown Trastuzumab, an antibody-based agent used for550HER2-positive breast cancer [147,148], and boronophenylalanine,551which is used for boron neutron capture therapy, can be delivered to552the brain and to brain tumor models [149,150]. FUS-induced BBB dis-553ruption has also been shown to improve the delivery of natural killer554cells in a brain tumor model [151]. Finally, a number of experiments555have loaded chemotherapy and other agents into the microbubbles556used for the disruption [146,152155], which offers the possibility of557achieving even higher local payload at the targeted region.558Delivering agents for neurodegenerative diseases, such asAlzheimer's,559Huntington's, and Parkinson's disease, have also been an active area of560research by several groups. A number of therapies for neurodegenera-561tive diseases such as neuroprotective agents [153,156], antibodies562[157,158], plasmidDNA [154], and siRNA [135] have all been successfully563delivered across the BBB using FUS and microbubbles. Other investiga-564tions have shown that circulating neural progenitor cells [159] or viral565vectors for gene therapy [160162] can be delivered to the sonicated566

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cross the BBB have been delivered to the brain or to brain tumormodelswith FUS and microbubbles. The amount of substance delivered andthe distance from the blood vessels that it penetrates appears to dependon its size. This is evident in the examples shown in Fig. 2, where lessdelivery of an albumin-bound MRI contrast agent (MW: ~67 kDa) wasevident compared to a standard agent (MW: 928 Da) in a macaque.This is even more clear in the example shown in Fig. 6, where deliveryof uorescent dextrans with different molecular weights was examinedafter sonication in the mouse hippocampus. For 3000 Da dextrans, arelatively uniform uorescence was observed; for the larger 70 kDa trac-er, itwasmore concentrated near the blood vessels, and a 2000 kDawasfound not to penetrate at all [136]. This result points to a need for closeexamination of how the delivery of large agents occurs it may not beenough to look for the presence of the agent, but to also investigatewhether it is delivered far enough from the vasculature at a highenough concentration to reach the desired target at a therapeuticlevel. Low-resolution methods such as MRI may not be sufcient forthis purpose. It may be possible, for example, for agents to make itpast the endothelial cells but get trapped at the basement membrane[137].

4.5.1. Delivery of therapeuticsA large number of therapeutic agents have also been delivered to the

brain and to brain tumor models (Table 4). Many of the studies so farhave investigated the delivery of chemotherapy agents, such as BCNU[138], doxorubicin [96], methotrexate [139], cytarabine [140], andtemozolomide [141]. Enhanced delivery of chemotherapy packaged inliposomes [83,142], targeted liposomes [143] and magnetic particles[144146], which allow for MRI-based tracking and enhanced deliveryvia magnetic targeting, have also been demonstrated (Fig. 7). Other

Table 3Example different tracers that have been delivered across the BBB.

Agent S

Lanthanum chloride 199mTc-diethylenetriaminepentaacetic pentaacetate 4Omniscan (Gd-DTPA-BMA) 5Magnevist (Gd-DTPA) 9Trypan blue, Evans blue ~Ablavar (gadofosveset trisodium) ~Horseradish peroxidase 4Dextran 3Immunoglobulin G ~pCMV-EGFPa ?MION-47 2Gold nanoparticles 5Gold nanorods 1Dotarem, P846, P792, P904, P03680 1

a Loaded into a microbubble.


ROOregions after FUS-induced BBB disruption. An example of delivery ofadeno-associated virus serotype 9 via FUS-induced BBB disruption to

the different cell populations in the mouse brain is shown in Fig. 8.

4.5.2. Disease modelsWhile delivery of these agents is promising, one also needs to dem-

onstrate that the amount of drug delivered and the drug penetration is sufcient to produce a therapeutic response. In some cases it is alsoimportant to demonstrate that the drug reaches the desired target andis active after it is delivered [156]. Several studies have shown thatFUS enhancement of the BTB can slow tumor growth and/or improvesurvival in orthotopic murine models of primary or metastatic braintumors [138,141,142,144,145,148,163].While in some cases the responsehas beenmodest, several of these studies have seen substantial improve-ments. Using multiple treatments may be necessary to achieve a pro-nounced improvement [142]. One factor that has not been investigatedin depth so far is to conrm that drugs can successfully be delivered toinltrating tumor cells, which are a major feature in glioma and otherprimary tumors, and to metastatic seeds. Both can be protected bythe normal BBB. The orthotopic models investigated so far do generallynot have large inltrating zones, and the benet observed in studies sofar may have been primarily due to FUS-enhanced permeability of theBTB. It may be challenging to get therapeutic levels to distant regionsthat are protected by the BBB. Some agents may have neurotoxic effectson the normal brain that may limit this ability.

Beyond brain tumors, a study by Jordo et al. showed that delivery ofantibodies targeted to amyloid plaques can reduce the plaque burden inAlzheimer's disease model mice [158]. While the decrease was modest,with multiple treatment sessions this may be an effective treatmentstrategy. In an intriguing follow-up study, the same group recently

Use

Da Electron microscopy tracer [109]Da SPECT agent [190]Da MRI contrast agent [89]Da MRI contrast agent [77]kDa Tissue dyes (binds to albumin) [79,191]kDa MRI contrast agent (binds to albumin) [79]Da Electron microscopy tracer [78]kDa Fluorescent tracer [136]kDa Endogenous antibodies [157]

Plasmid DNA [192]m MRI contrast agent [120]m Carrier for drugs or imaging [193]40 nm Photoacoustic imaging contrast agent [194]nm MRI contrast agents [115]in barrier disruption for targeted drug delivery in the central nervous4.01.008

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597 [164]. We anticipate that these studies are only the beginning, and598 that FUS has a large potential for Alzheimer's disease and other neuro-599

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t4:4 Table 4t4:4 Example therapeutic agents that have been delivered across the BBB or BTB.

Therapeutic agent Size Use Delivered tot4:4

Temozolomide 194 Da Chemotherapy Glioma model (9L)a [141]t4:41,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)b 214 Da Chemotherapy Glioma model (C6)a [138,152]t4:4Cytarabine 243 Da Chemotherapy Normal brain [140]t4:4Boronophenylalanine 330 Da Agent for boron neutron capture therapy Glioma models (GBM 8401 [149]; 9L [150])t4:4Doxorubicin 540 Da Chemotherapy Normal brain [96]t4:4Methotrexate 545 Da Chemotherapy Normal brain [139]t4:4siRNA ~13 kDa Huntington's disease therapy Normal brain [135]t4:4Glial cell line-derived neurotropic factor (GDNF)b 24 kDa Neuroprotective agent Normal brain [153]t4:4Brain-derived neurotropic factor (BDNF) 27 kDa Neuroprotective agent Normal brainc [156]t4:4Herceptin (trastuzumab) 148 kDa Anti-cancer antibody Normal brain [147]; breast cancer brain met. model (BT474)a [148]t4:4BAM-10 A targeted antibodies ~150 kDa Therapeutic antibody for Alzheimer's disease TgCcrND8 Alzheimer's model micea [158]t4:4BCNU-VEGFb ~150 kDa Antiangiogenic-targeted chemotherapy Glioma model (C6)a [155]t4:4Plasmid DNA (pBDNF-EGFP)b ~3600 kDad Gene therapy Normal brain [154]t4:4Epirubicin in magnetic nanoparticles ~12 nm Magnetic targeted chemotherapy Glioma model (C6)a [144]t4:4Doxorubicin in magnetic nanoparticlesb ~610 nm Magnetic targeted chemotherapy Glioma model (C6) [146]t4:4

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degenerative disorders. Issues regarding the feasibility and safety ofdisrupting the BBB in large brain regions (or the whole brain perhapsrepeatedly) may be need further investigation, however.

4.6. Methods to plan, monitor, and evaluate FUS-induced BBB disruptionshowed that FUS-induced BBB disruption alone can reduce the size ofthe plaques, perhaps through the delivery of endogenous antibodies

BCNU in magnetic nanoparticles ~1020 nm Magnetic targeted cheAdeno-associated virus (AAV) ~25 nm Gene therapy vectorLiposomal doxorubicin (Lipo-DOX) 90 nm ChemotherapyInterleukin-4 receptor targeted Lipo-DOX 100

120 nmChemotherapy

Neural progenitor cells 710 m Stem cellNatural killer cells (NK-92) ~10 m Cell therapy for brain

a Also showed improved outcomes with FUS-induced BBB disruption.b Used drug-loaded microbubbles.c Also showed drug activity after delivery.d Assumed 660 Da per base pair (bp), 760 bp for BDNF, and 4700 bp for pEGFP-N1.UNCORR

As described above, FUS-induced BBB disruption utilizes themechanical interactions between microbubbles oscillating in the ultra-sound eld and the vasculature. These interactions critically dependon the exposure parameters as well as the vascular density and perhapsother properties of the vascular bed. The latter, can affect the localconcentration of microbubbles, how they interact with the ultrasoundeld [114], and, more importantly, how much drug will be deliveredto the brain [79]. Unfortunately, many of these parameters are difcultto predict and are expected to vary signicantly across different patientsand diseases. Thus, methods are needed to (i) determine what parame-ters to use (treatment planning), (ii) rene them during sonication to

Fig. 6. Delivery of uorescent dextrans with different sizes across the BBB in a mouse using FUgions can be observed for all dextrans, whereas spots of high uorescence are observed only wModied from Proc Natl Acad Sci U S A 2011; 108: 1653916544; 2011 by The National Aca


ROO

ensure BBB disruption without overexposure (treatment monitoring),and (iii) evaluate the treatment effects (treatment evaluation).

4.6.1. Treatment planningInmost cases, experiments evaluating FUS-induced BBBdisruption in

animal models have used a xed set of acoustic parameters determinedfrom prior experience and simple, geometrically-focused transducers. Ingeneral, accurate targeting can be achieved with such systems usingstereotactic frames [165] if image-guidance is not available, and fairlyrepeatable results can be obtained with sonication through the thinskull inmice and rats, or in larger animals through a craniotomy.Methods

herapy Glioma model (C6)a [145]Normal brain [160162]Normal brain [83]; Glioma model (9 L)a [142,163]Glioma model (8401) [143]

Normal brain [159]or Breast cancer brain met. model (MDA-MB-231-HER2) [151]624to avoid standingwaves [81] and that take into account variations in skull625thickness [166] can improve repeatability in small animal studies where626transcranial sonication is used.627Such approachesmay be challenging to translate to human subjects,628where the thicker skull is complex (a layer of trabecular bone629surrounded by layers of cortical bone) and can vary substantially630between individuals (3.59.5 mm [167]). The skull, which has a sub-631stantially higher acoustic impedance than soft tissue, will reect most632of the ultrasound beam, and the amount transmitted will depend633strongly on the angle between the bone and the face of the transducer634[168]. Its irregular shape can also deect and distort the beam, and

S and microbubbles (A: 3 kDa; B: 10 kDa; C: 70 kDa; bar: 1 mm). Diffuse uorescence re-ith the 70-kDa dextran.demy of Sciences of the USA.


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reections within the skull cavity need to be taken into account. Tocorrect for beam-aberrations introduced by thick skulls, phased arrayscomposed of more than 1000 elements combined with skull aberrationcorrection algorithms that utilize CT data are employed [57,58]. Thesearrays can also be programmed to rapidly steer the beam electronicallytomultiple targets enabling coverage of tissue volumes [79], anddifferentportions of the array can be disabled to reduce internal reections orexclude certain structures.

While these approaches that use acoustic simulations and CT scansare effective in restoring the focusing of the array after transmissionthrough the skull, clinical experience with them for thermal ablationhave shown that one still needs to correct for small errors (~12 mm)in targeting [60,63]. To achieve this correction, one needs to be able tovisualize the focal region at exposure levels that do not induce damageor other unwanted effects. Currently, this can be achieved using MRI-based methods that can visualize low-level (12 C) focal heating[59,169] or map small tissue displacements of a few microns inducedby radiation force [170].

Ensuring accurate targeting will be most important if one aims toprecisely disrupt the BBB at discrete locations. In addition, since thestrength of the total microbubble activity, as well as magnitude of thedisruption will depend on the vascularity of the targeted tissue (grayvs. whitematter, for example [79]), it will be important to know exactlywhere the target is located. It might also be desirable to avoid directsonication on large blood vessels. If one is uncertain about the targetingof the focal region, it may be challenging to understand whether a pooror unexpected result is due to an incorrect exposure level or tomistargeting. Pre-treatment imaging delineating vascularity, perfusion,

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Fig. 7. Confocal micrographs of tissue from tumor after FUS-induced BTB permeabilizationfollowed by magnetic targeting and contralateral brain regions after delivery of magneticnanoparticles (MNPs) loaded with epirubicin. Dark structures in the phase micrographsshow MNPs (left); fused uorescence images (right) indicate the presence of epirubicin(red) and DAPI-stained nuclei (blue). Arrows indicate the capillaries; epirubicin occursin the capillary beds but does not penetrate into the brain parenchyma. (For interpretationof the references to color in this gure legend, the reader is referred to the web version ofthis article.)Modied from Proc Natl Acad Sci U S A 2010; 107: 1520515210; 2011 by The NationalAcademy of Sciences of the USA.


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or other vascular properties may prove useful for planning the treat-ment. Itmay also be useful to combine thesemeasurementswithmodelsof the microbubble oscillations within the microvasculature [114,171].

Accurate control of the focal pressure is critical to ensure BBBdisrup-tion is produced while preventing inertial cavitation. The thick andcomplex human skull makes accurate focal pressure estimationsextremely challenging.While the acoustic modelingmethods developedfor aberration correction may provide estimates of the focal pressureamplitude, it has not been validated to our knowledge. Itmay be possibleto use the MRI-based methods mentioned above that can visualize focaldisplacements or heating to ensure a predictable focal pressure ampli-tude. Marty et al., for example, usedMR acoustic radiation force imagingto ensure a consistent exposure level between subjects in BBB disruptionexperiments in rats [115]. However, one needs to take the underlyingtissue properties (which may be unknown for tumors or other abnor-malities) into account or test it in proximal normal brain locations.

4.6.2. Treatment monitoring and controlGiven the challenges in predicting the focal pressure amplitude

when sonicating transcranially, we anticipate that effective monitoringof the procedure will be important if this technology is to be translatedto clinical use. Atminimum, suchmonitoring should provide an indicationthat the exposure level is sufcient to induce BBB disruption and alertthe user if inertial cavitation is occurring. One could use MRI methodsfor this purpose. Contrast-enhanced imaging can be used to visualizewhen the disruption occurs, and T2*-weighted or susceptibility-weighted MRI can be used to detect petechiae produced by inertialcavitation [77,119]. These methods could be used now for control overthe procedure in initial clinical tests of FUS-induced BBB disruptionwith experienced users. However, performing multiple MRI acquisi-tions would be time-consuming and might require excessive amountsof both ultrasound and MRI contrast agents. Real-time and, perhaps,more direct methods are likely necessary for widespread clinicalimplementation.

For real-time monitoring and control, a number of studies haveinvestigated the use of piezoelectric receivers operated in passivemode (i.e. only listening) to record and analyze the diverging pressurewaves (i.e. acoustic emissions) emitted by oscillating microbubblesduring FUS-induced BBB disruption [105107,172,173]. The spectralcontent and strength of the recorded emissions is sufcient to charac-terize and subsequently control the microbubble oscillations. Inertialcavitation is manifested in the frequency domain of the acoustic emis-sion as a broadband signal [53], and has generally been associated withthe production of vascular damage during BBB disruption [105,107],although other studies have suggested that it can occur without damage[106]. Harmonic and/or sub- and ultra-harmonic acoustic emissions inthe absence of broadband signal are indicative of stable volumetricoscillations, which consistently have been associated with safe BBB dis-ruption [105107]. Therefore, depending on the spectral content andstrength of the emissions the output of the device can be increaseduntil strong harmonic, subharmonic, or ultraharmonic emissions areobserved, and decreased if broadband emissions are detected. O'Reillyet al. demonstrated a closed-loop controller built around the detectionof ultraharmonic emissions to automatically select an acoustic exposurethat could produce BBB disruption with little or no petechiae [173]. Wehave been exploring the strength of the harmonic emissions as a basisfor such a controller, as we have found that we can reliably detect itbefore inertial cavitation occurs and that it is correlated with themagni-tude of the BBB disruption measured via MRI contrast enhancement[105,107]. An example of this correlation observed during transcranialBBB disruption in macaques using a clinical brain FUS system is shownin Fig. 9A.

If one can integrate a large number of receivers into the FUS system,one can use passive reconstructionmethods [174,175] to create two- oreven three-dimensionalmaps of themicrobubble activity to ensure that727it is occurring at the expected location. Examples from experiments in


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our laboratory in macaques using a linear receiver array integrated intoa clinical brain FUS system are shown in Fig. 9BC. In these experiments,we found that the cavitation activity in the passive acoustic maps (redarea in Fig. 9BC) was co-localized with the resulting BBB disruption[176].

4.6.3. Treatment evaluationAs described above, contrast-enhanced imaging and T2*- or

susceptibility-weighted imaging can be used to verify that BBB disrup-tion has occurred and whether signicant vascular damage hasoccurred, respectively. For tumors, it may be necessary to compare thesignal enhancement after contrast injection to measurements obtainedbefore FUS. Other imaging modalities may also be useful [177]. If thecontrast-enhanced imaging is obtained before the therapeutic agent isinjected, one can conrm that the BBB disruption is only occurring atthe targeted locations before administering the drug, providing anotherlevel of control to ensure that drugs are delivered only to desiredregions.

Post-treatment imaging could be more useful if one could use it toestimate drug uptake and penetration in the brain. This can be achieveddirectly by labeling the drug with a contrast agent for MRI or othermodality [146]. It might also be possible to use a standard contrastagent as a surrogate measurement. A number of studies have relatedsignal intensity changes of contrast-enhancedMRI at the end of the son-ication with tissue drug concentrations [83,147,178]. More quantitativeand repeatable techniques, such as estimating contrast agent concentra-tions via T1-mapping [115,179] or vascular transfer coefcients viaanalysis of dynamic contrast-enhanced MRI (DCE-MRI) [180] have

Fig. 8. Gene transfer to neurons, astrocytes, and oligodendrocytes after delivery of adeno-assovia FUS-induced BBB disruption. Immunohistochemistry was used to detect GFP expression(B) GFAP-positive cells (astrocytes, white arrows) and (C) Olig2-positive cells (oligodendrocytModied from Human Gene Therapy 23:11441155 (November 2012); 2012 Mary Ann Lieb

Please cite this article as: M. Aryal, et al., Ultrasound-mediated bloodbrasystem, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/j.addr.201 PROOFED

755been used to perform spatial and temporal characterization of BBB per-756meability. DCE-MRI can also predict the resulting payload of drugs to757the brain [96] and in some cases, in tumors [179]. Examples showing758DCE-MRI evaluation of BBB disruption and its subsequent restoration759over time, and its relationship to concentrations of doxorubicin are760shown in Fig. 10. If one understands the relationship between the con-761centrations of the therapeutic and the imaging contrast agent, which762can perhaps be established in animals, one might be able to titrate the763drug administration to achieve a desired level in the brain. However,764this may be challenging in tumors, where the vascular permeability765can change over time [179].

7665. Going forward

767Based on the extensive preclinical experiencedescribed above, along768with recent studies in non-human primates [79,80] demonstrating that769themethod can be scaled upwithout producing evident tissue damage770or functional decits even after repeated sessions [79], this method for771targeted drug delivery in the brain is ready in our view for initial safety772tests in humans, where it will hopefully reveal its enormous potential.773Clinical transcranial MRI-guided FUS systems [60,63] and commercially-774available ultrasound contrast agents are available and can be used for775these tests. Given the huge clinical need and the existence of available776approved anticancer agents that are expected to be effective if they777could be adequately delivered, brain tumors may be an appropriate778target for these initial tests.779Given MRI's high cost and complexity, coupled with the need in780many cases to administer therapeutics over multiple sessions, it would

ciated virus serotype 9 carrying the green uorescent protein (GFP) to the mouse brainin hippocampus for (A) NeuN-positive cells (neurons, white arrows), and striatum fores, white arrow).ert, Inc.


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be desirable in the long run to develop systems to provide FUS-inducedBBB outside of theMRI environment. Passive cavitationmonitoring and/or mapping may be the enabling technology for this translation awayfrom MRI guidance. One can envision systems that use pre-treatmentMRI and CT (to delineate different tissue structures and skull density,respectively), along with frameless navigation and cavitation moni-toring to provide routine BBB disruption in an outpatient facility. Anumber of technical developments, such as reducing targeting errorduring transcranial sonication, nding methods to easily register theposition of the skull within the FUS device without a stereotacticframe, and developing methods to better quantify acoustic emissionsmeasurements obtained through the thicker human skull, are neededto reach this goal. In our opinion, all of these things are achievable. Itwould also be desirable to remove the need to shave the head, whichis currently needed to allow for acoustic coupling. Attenuation shouldnot prevent this [181].

The potential of this technique to manipulate the amount of drugdelivered to each point in the brain can provide a level of control thatis not readily available with existing technologies. This control can beachieved by modulating the acoustic parameters to control the level

Fig. 9. Comparison of MRI signal enhancement with microbubble acoustic emissions.(A)MRI signal enhancement afterGd-DTPA injection plotted as a function of theharmonicemissions signal strength measured with single-element detectors. A clear relationshipbetween the two measurements was observed with a good t to an exponential. Theagreement appeared to hold among different animals and in cases where targets withlow-level harmonic emissions were sonicated a second time with either a higher powerlevel or an increased dose of microbubbles. Reprinted from PLoS ONE 7(9): e45783.http://dx.doi.org/10.1371/journal.pone.0045783; 2012 Arvanitis et al. (BC) Comparisonbetween MRI signal enhancement and passive cavitation mapping. (B) Map showing theenhancement relative to a pre-contrast image. (C) Fusion of passive acoustic map withthe T1-weighted MRI from (B). The red region shows the pixels in the cavitation mapwithin 95% of themaximumvalue. This region overlappedwith the contrast enhancement.The pixelwith themaximumcavitation activity is notedwith a +. The enhancement fromother targets sonicated in the same session is visible. (For interpretation of the references tocolor in this gure legend, the reader is referred to the web version of this article.)Modied from Phys Med Biol 58.14 (2013): 474961; 2013 Institute of Physics andEngineering in Medicine.


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801of the disruption, by analyzing the post-sonication contrast enhancement802before injecting the drug and titrating the drug dose, or by repeating803sonication at select areas after some delay to lengthen the time the bar-804rier is disrupted [96]. It might even be possible to tailor the sonication805parameters and BBB disruption to the molecular weight of the thera-806peutic agent. These methods may enable dose painting that can give807clinicians new exibility in how drugs are used in the CNS to maximize808efcacy and minimizing side effects. Further targeting and control can809be achieved by loading drugs into microbubbles [146,152155] or by810using magnetic targeting after FUS-induced BBB disruption [144146].811It will also be important to establish the feasibility and safety of812targeting very large volumes. Many promising applications of this tech-813nology (brain tumors, Alzheimer's disease, etc.) will require sonication814over large portions of the brain for greatest effect. This can be achieved

Fig. 10.UsingDCE-MRI to evaluate FUS-BBB disruption. (AB)Mean Ktrans valuesmeasuredvs. time from DCE-MRI in regions of interest at sonicated locations and in correspondingnon-sonicated structures in the contralateral hemisphere. The decay of K, which occurreddue to restoration of the BBB, was t to an exponential decay (solid line; dotted lines:95% CI). (A) Decay after a single sonication. (B) Time course after two sonications separatedby 120 min. The second sonication increased the amount of time the barrier was disrupted.Values were signicantly higher than in control locations in the contralateral hemisphere(**P b 0.01 and *P b 0.05). (C) DOX concentration achieved at the sonicated locations asfunction of Ktrans measured using DCE-MRI 30 min after sonication. The DOX concentrationwas measured approximately 16 h later for brain targets in single sonication (SS), singlesonication with hyperintense spots in T2* weighted image (SS (+T2*)), double sonicationwith 10 min interval (DS 10 min), and double sonication with 120 min interval (DS120 min). The solid line shows a linear regression of the data (slope: 28,824 ng/g DOXper change in Ktrans in min1; intercept: 377 ng/g DOX; R: 0.7).Modied from Journal of Controlled Release 2012;162:134142; 2012 Elsevier B.V.


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829 disruption were elucidated. Without knowledge of the mechanism,830 one can only speculate on how one can optimize the procedure, and831

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13M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014) xxxxxxUNCORREC

we are left with performing time-consuming parametric studies. Giventhe large parameter space in variables that can inuence the magnitudeof the BBB disruption, it is possible that we have not stumbled uponparameters that can further improve upon the safe window where BBBdisruption is possible. Multidisciplinary approaches are very likely toprove fruitful and could potentially identify a unique physiologic mecha-nism, perhapswith interesting implications on the structure and functionof the vasculature in the CNS.

Other tissues have barriers similar to the BBB and could benet fromsimilarmicrobubble-enhanced sonications or prove to be simplermodelsto study the aforementioned interactions. There is data demonstratingthat disruption of the bloodretinal [182] and bloodspinal cord [183]barriers can be disrupted by FUS, and that the glomerular function inthe kidney can be enhanced, presumably through changes in thebloodurine barrier [184]. Also, using pressure amplitudes higher thanare needed for FUS-induced BBB disruption, one can use microbubble-enhanced sonications for ablation [185], thrombolysis [186], orradiosensitization [187]. One can potentially combine one of thesemicrobubble-enhanced therapieswith BBBdisruption to produce a syn-ergistic effect or to deliver therapeutics in the surrounding tissues.

The clinical need for new approaches to bypass the BBB in order toincrease the number of drugs that can be used effectively in the CNS istremendous. In addition to CNS disorders such as brain tumors, stroke,trauma, and genetic neurodegenerative disorders, opportunities mayexist for awide range of other applications. Examples include painman-agement, and psychological disorders such as addiction, both of whichmay benet from a technology that permits drug transport to precisetargets in the brain. Existing drugs for these conditions can have severeside effects that limit their use. With an ever-growing knowledge ofbrain function and dysfunction, precise drug targeting in the CNS mayprove to be particularly important. Technical improvements that areachievable in our view could enable FUS to be used on a wide scale forroutine targeted drug delivery to the CNS.

6. Conclusion

FUS is a unique technology that can induce BBB or BTB perme-abilization that is targeted, noninvasive and transient. Extensive workin preclinical studies has demonstrated that it can enable the deliveryof therapeutics that normally do not reach the brain, and enhancetheir delivery to brain tumors. The sonications do not appear to haveanydeleterious effects on the brain, and themethod is readily repeatable.MRI and acoustic methods to plan, monitor, and evaluate the treatmentoffer the possibility of having control over where drugs are deliveredand in what concentration. Given the availability of clinical FUS devicescapable of focusing ultrasound through the intact human skull, alongwith recent safety studies demonstrating the method can be performedsafely in nonhuman primates, it appears that the method is ready forby systematically focusing the ultrasound beam to a large number ofindividual targets. It should be possible to target the hundreds or thou-sands of focal points needed to achieve such large-scale BBB disruptionin a reasonable amount of time. Given the low duty cycle and minimalacoustic exposure levels needed to induce the effect, many targets canbe sonicated simultaneously. For example, with the low duty cycle need-ed to induce BBB disruption (1% or less), we can target 100 targets ormore with electronic beam steering with a phased array in the sameamount of time it currently takes to disrupt one location with the simplesystemweuse for small animal experiments.While again thiswill requiretechnical improvements and more safety tests, we expect that achievingcontrolled, large volume BBB disruption is achievable.

It would be helpful if the physical and/or physiological mechanismsby which the mechanical effects of FUS and microbubbles induce BBBinitial clinical tests.


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References

[1] E.A. Neuwelt, B. Bauer, C. Fahlke, G. Fricker, C. Iadecola, D. Janigro, L. Leybaert, Z.Molnar, M.E. O'Donnell, J.T. Povlishock, N.R. Saunders, F. Sharp, D. Stanimirovic,R.J. Watts,

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