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Advanced Drug Delivery Reviews xxx (2014) xxxxxx
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ADR-12569; No of Pages 16
Contents lists available at ScienceDirect
Advanced Drug D
j ourna l homepage: www.eUN4.6.3. Treatment evaluation . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. .
5. Going forward . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . .6.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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. . . . . . . . . . . . . . . . . .CO4.5. Delivery of
imaging/therapeutic agents and tests in animal disease models . . .
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4.5.1. Delivery of therapeutics . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
04.5.2. Disease models . . . . . . . . . . . . . . . . . . . . . .
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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 .
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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. . . . . . . . . . . . . . .
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0iopharmaceutical approaches . . . . . . . . . . . . . . . . . . .
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0Contents T
ED P
ROOF
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
1CA174645, P41EB015898,
A 02115, USA. Tel.: +1 617
ld).
vier B.V.
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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t1:1
t1:1
t1:1
d; syt1:1
e, bt1:1t1:1
s; lat1:1
t1:1
g de; cas fort1:1
2 M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014)
xxxxxxUNCORREC
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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xxxxxxUNCORREC
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
ROOF
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
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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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,
in barrier disruption for targeted drug delivery in the central
nervous4.01.008
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361
362
363
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
378
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5M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014)
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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
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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
442
443
444
445
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
477
478
479
480
Q2 btaiforee tathed b0 m
6 M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014)
xxxxxxUNCORREC
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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OF
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Q3 art on. Thsonacesemn xNo pen. Snmwer20
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
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
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.
in barrier disruption for targeted drug delivery in the central
nervous4.01.008
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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
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
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545
546
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548
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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izet3:3
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t3:3
0 nt3:30 nt3:30 t3:365t3:3
t3:3
8 M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014)
xxxxxxUNCORREC
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.
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
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
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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
mott4:4t4:4
t4:4
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9M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014)
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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
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
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.
in barrier disruption for targeted drug delivery in the central
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10 M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014)
xxxxxxUNCORRECT
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.
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
ROOF
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
in barrier disruption for targeted drug delivery in the central
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11M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014)
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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.
in barrier disruption for targeted drug delivery in the central
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12 M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014)
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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.
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
RO
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.
in barrier disruption for targeted drug delivery in the central
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mechanism,830 one can only speculate on how one can optimize the
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13M. Aryal et al. / Advanced Drug Delivery Reviews xxx (2014)
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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.
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
ROOF
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