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Recent Advances in Transcranial Focused Ultrasound (FUS) Triggered Brain

Delivery

Article in Current Drug Targets · December 2016

DOI: 10.2174/1389450117666161222160025

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Current Drug Targets, 2017, 18, 000-000 1

REVIEW ARTICLE

Recent Advances in Transcranial Focused Ultrasound (FUS) Triggered

Brain Delivery

Nidhi Nainwal*

Department of Pharmacy, GRD (PG) IMT, Rajpur Road, Dehradun, 248001 Uttarakhand, India

ARTICLE HISTORY
Received: November 30, 2015
Revised: May 17, 2016
Accepted: December 16, 2016
DOI:
10.2174/1389450118666170125144557
Abstract: Successful delivery of therapeutic agents in the brain is very challenging, which makes
treatment of various brain disorders very difficult. One of the major causes of this difficulty is the
presence of physiological barriers in the brain like blood brain barrier (BBB) and blood cerebro-
spinal fluid (CSF) barrier. These barriers are very specific and restrict the entry of water soluble
agents and ionized molecule. Only lipid soluble and small molecular size agents are allowed to enter
into the brain. But there are several serious brain diseases in which the use of only conventional small
molecular mass lipophilic drug is not effective. Various approaches like the use of BBB permeation
enhancers, osmotic disruption of BBB by using mannitol, prodrug approach, etc. have been developed
for the transportation of drug across the brain. The current article focuses on the transcranial route,
transcranial focused ultrasound (FUS), the techniques involved in transcranial focused ultrasound
(FUS) delivery and recent updates of this system.

Keywords: Blood brain barrier, transcranial delivery, focused ultrasound.

1. INTRODUCTION drug molecule known as lipidization,” to enhance penetra-

1.1. Blood Brain Barrier
Blood brain barrier (BBB) was discovered by Paul Ehr-
lich. The BBB protects the brain from its environment by
restricting the entry of unwanted toxic substances, various
microorganisms into the brain [1, 2]. The BBB is formed by
tight junctions between adjacent endothelial cells, and thus
BBB restricts the permeation of drugs that have larger mo-
lecular size, hydrophilic in nature and is present in the ion-
ized form. The barrier allows diffusion of small, highly lipo-
philic agents. This barrier is so effective that only about 2%
of all small molecules (<600 Dalton) are allowed through
and none of the larger ones [3]. To allow the diffusion or
transport of larger drug molecules or diagnostic agents into
the brain, it is, therefore, essential to temporarily disrupt the
BBB.
1.2. Transcranial Delivery
Large molecular weight therapeutic substances or drugs
such as monoclonal antibodies, proteins, and gene therapeu-
tics etc. cannot penetrate the BBB. Therefore, it is crucial to
develop a technique that provides targeted delivery in the
brain without any side effects. One technique for brain deliv-
ery includes attachment of lipid groups to the hydrophilic
*Address correspondence to this author at the Department of Pharmacy,
GRD (PG) IMT, Rajpur Road, Dehradun, 248001 Uttarakhand, India; Tel:
+91-9411592536; E-mail: nidhi.nainwal87@gmail.com
tion of drugs. The drawback of the technique is that in ad-
dition to the delivery in the desired area of the brain, the
drug is permeable in the entire brain and body that causes
unwanted side effects [3]. Another technique includes the
use of penetration enhancers, osmotic disruption of the
brain, the use of carriers, etc. New developments have
shown the transcranial method as a promising technique for
brain targeting. Transcranial means passing or performed
through the skull or cranium. Transcranial drug delivery
route is used for the treatment of the various Central nerv-
ous system (CNS) disorders, which allows small molecules
to traverse the blood brain barrier across the skull bone to
reach the target site [4, 5]. Transcranial delivery is there-
fore potentially applicable to drugs failed in the develop-
ment stage due to poor BBB penetration and also reduces
the off-target side effects associated with peripheral drug
administration.
2. TRANSCRANIAL FOCUSED ULTRA SOUND (FUS)
Transcranial focused ultrasound penetrate the skull. Fo-
cused ultrasound (FUS) is a technique that temporally dis-
rupts the BBB and thus increases the permeability of thera-
peutic agents across the brain [6] (Fig. 1).
In a study [6] the phenomenon of focused ultrasound
(FUS) mediated opening of the BBB was observed in the
presence of microbubbles, under the monitoring through
Magnetic resonance imaging and Magnetic resonance con-
trast agents.

1389-4501/17 $58.00+.00 © 2017 Bentham Science Publishers

2 Current Drug Targets, 2017, Vol. 18, No. 10
Fig. (1). Opening up the BBB to deliver drugs. (1) The BBB is impermeable to most of the drugs (2). Focused ultrasound can reversibly open
the BBB, allowing the drug to enter the brain that normally could not.
Choi et al. [7] investigated FUS mediated disruption of
the BBB in the hippocampus of Alzheimer′s model mice
through the intact skull and skin. The result showed high
localized permeability and retention in the brain [8-11]. Ul-
trasound has the advantage of improving brain delivery of
therapeutic agents with the use of micro carrier, nanoparticle,
liposomes and also improves the transport through the ex-
tracellular matrix, and facilitate the cellular uptake. The ac-
tion may be due to either thermal or non-thermal effects. The
nonthermal action involves cavitation and radiation force.
Vykhodtseva et al. [12] first observed the ultrasound trig-
gered BBB opening in the rabbit brain. Focused ultrasound is
an operator-dependent technique. For this reason, the repro-
ducibility of the ultrasound technique has been evaluated by
Marco Matteo Ciccone et al. [13] in Multiple sclerosis (MS)
patients. They tried to compare the values coming from two
experienced operators who evaluated 32 consecutive patients
independently from each other to calculate interobserver
variability. The McNemar test confirmed the procedure re-
producibility between two operators with no statistical dif-
ferences between themselves.
2.1. Transcranial Ultra Sound (US) Mediated
Sonothrombolysis
Sonothrombolysis is a novel treatment for acute intracra-
nial arterial occlusion. Improved recanalization has been
found with “diagnostic” transcranial ultrasound (US) in
combination with standard intravenous thrombolysis with
recombinant tissue-plasminogen activator (rtPA) in two ran-
domized trials [14, 15]. Truebestein et al. [16] for the first
time demonstrated that high-frequency ultrasound can be
used to dissolve thrombi. Alexandrov et al. [14] confirmed
the potential of ultrasound-mediated thrombolysis. This
treatment method may be used as an alternative, in the situa-
tion when the use of thrombolytic drugs or thrombus extrac-
tion is contraindicated [17, 18]. Microspheres-mediated
Nidhi Nainwal
thrombolysis for successful drug delivery and improvement
of microcirculation has been developed [19]. Recanalization
using intra-arterial high-energy US catheter has been tested
in a recent pilot study [20].
2.2. Transcranial Ultrasound and Microspheres Medi-
ated Drug Delivery in Thrombolysis
The combination of transcranial US and microspheres
may facilitate the delivery of drugs across the BBB. Micro-
spheres have both capabilities, to improve diagnostic images
and to mechanically agitate fluid that potentiates thromboly-
sis and thus may help in targeted drug delivery. This dose-
escalation study of microspheres showed increased bleeding
in the second dose tier, prompting the sponsor of the study to
discontinue this approach. Microspheres with abciximab are
used for better thrombus binding and identification and im-
proved sonothrombolysis. In vivo, molecular imaging of the
human thrombus can be carried out with microspheres con-
jugated with abciximab, a glycoprotein IIb/IIIa receptor in-
hibitor that is involved in ligand targeting of the thrombus.
In vitro experiments have shown that improved binding of
microspheres to the clot enhances sonothrombolysis [21, 22].
Systemic administration of tissue plasminogen activator
(tPA) is the fastest way to start the treatment of acute
ischemic stroke. In a recent feasibility study addition of
gaseous perflutren lipid microspheres to tPA and transcranial
Doppler further facilitated early flow improvement, with a
50% rate of early, complete recanalization [23-25]. Recent
promising human studies with transcranial Doppler (TCD)
enhanced thrombolysis included gaseous microspheres as an
adjunctive facilitator of thrombus breakup with a systemic
tissue plasminogen activator (tPA) therapy [26]. After a
gaseous microsphere is compressed by ultrasonic mechanical
pressure waves, its shell may break up the microsphere ex-
pand in size and become “activated”. Under low diagnostic
ultrasound pressures, microsphere can also oscillate without
 Transcranial Focused Ultrasound (FUS) Triggered Brain Delivery
shell break-up. In comparison to red blood cells micro-
spheres have much higher impedance and thus act like bright
reflectors by sending back stronger echoes useful for imag-
ing. With expansion microsphere also transmit mechanical
momentum to surrounding fluids potentially causing multi-
ple “micro angioplasties” to thrombus with the agitation of
stagnant residual flow. Because the process of activation
produces traceable echoes, quantification of appearance on
diagnostic ultrasound can aid dose calculations of micro-
sphere delivered to target tissues [27].
2.3. Microbubble Facilitated Focused Ultrasound (MB-
FUS) Induced BBB Opening
In comparison to the alternative brain drug delivery tech-
niques like hypertonic infusion [28] and modified lipophilic
chemical use. Microbubble facilitated focused ultrasound
(MB-FUS) is a noninvasive technique that causes local dis-
ruption of the BBB, increase the local concentration of vari-
ous therapeutic agents and reduces the risk of off-target side
effects. BBB is reversibly disrupted by this technique and
thus a time window of several hours is provided for drug
release in the brain [29-34]. Microbubbles (MBs), bubbles
with diameters of less than 10 µm, are usually made of a
phospholipid, surfactant, albumin, or synthetic polymer shell
filled with high molecular weight gas with a very low water
SF6, solubility (e.g., sulfurhexafluoride or perfluorepropane gas)
C3F8 [35]. Microbubbles are usually prepared by mechanical agi-
tation, sonication, or by the use of micro fluidic devices.
Microbubbles with a diameter of 1- 4 mm are generally used
for ultrasound contrast enhancement. Microbubbles were
initially developed as tools for ultrasound imaging. The first
microbubbles were microscopic spheres comprised of air and
had an extremely short half-life. Later on the air in the mi-
crobubbles was replaced with heavier fluorinated compounds
that are dissolved more slowly. Coated microbubbles pro-
vided a protective shell composed of protein, phospholipids,
or biocompatible polymers. Charged surfactants and Poly-
ethylene glycolated lipids were also incorporated into the
microbubbles to stabilize the shell and to prevent bubbles
coalescence while also prolonging the circulation time [36].
Phospholipid monolayer coating of microbubble increased
longevity without comprising on the acoustic response.
2.3.1. Microbubbles as Ultrasound Contrast Agents
In 1968 the use of microbubbles in echocardiography
was reported [37]. MBs can be used in routine examinations
of organ perfusion, highly vascularized tumor structures, in
the diagnosis of kidney and heart disease [38-41]. Opti-
sonTM (GE Healthcare, WI, USA) [42, 43], De-
finity® (Lantheus Medical Imaging, MA, USA) and
SonoVue® (Bracco, Milano, Italy) are the commercial MB
agents that are approved for clinical examination with size
larger than 1 µm and imaging durations of 5-10 min [44-46].
2.3.2. MB- FUS Mediated Delivery to the Central Nervous
System (CNS)
In addition to ultrasound contrast agent for diagnostic
applications, MBs also has unique therapeutic applications
[47-49]. Bioactive substances such as genes, drugs, proteins,
etc. can be delivered by MBs. Various approaches have been
Current Drug Targets, 2017, Vol. 18, No. 10 3
suggested for incorporation of therapeutic agents in MB car-
riers [50, 51], e.g., attachment to the outer shell surface, em-
bedding within the shell, dissolving hydrophobic drugs in the
oily layer between the gas core and shell, and linking them to
the shell, for example via streptavidin biotin interactions [52,
53]. Carriers such as liposomes, micelles, or microspheres
can be used for drug loading and these structures can then be
easily attached to lipid MBs, usually via avidin-biotin inter-
actions [54]. Both hydrophilic and hydrophobic drugs can be
encapsulated in these types of MB complexes. Combined use
of microbubble with focused ultrasound is a noninvasive
method that results in reversible disruption of BBB locally
rather than systemically. Therapeutic agents are delivered
across the BBB by passive diffusion. SonoVue®, which con-
sists of sulfur hexafluoride-filled microbubbles of phosphol-
ipids, and Levovist®, granulates of galactose and palmitic
acid, which binds to micrometer-sized air bubbles are com-
mercially available. Following IV injection, they take energy
on under the influence of the US, and by oscillation or rup-
ture, this energy is released again, which reinforces the US
effectiveness. Various experiments have shown the effec-
tiveness of this method without an increase in the intracranial
bleeding rate, which has been demonstrated in vivo. Molina
et al. [26] showed an improvement by an intermittent bolus
injection of Levovist® in addition to tPA treatment plus 2-h
insonation with TCD monitoring. A similar study was con-
ducted by Perren et al. [55] in which patients who had suf-
fered from a MCA stroke underwent IV rtPA thrombolysis
and 2-MHz TCCS monitoring for 1 h with SonoVue®, result-
ing in a clinical improvement in these patients. No additional
intracranial bleedings were noted in these studies.
2.3.3. Transcranial Ultrasound and Microbubble Mediated
Drug Delivery in Thrombolysis
Tachibana et al. [56] demonstrated ex vivo that the co
administration of microbubbles improves the thrombolytic
activity of US in combination with pharmaceutical throm-
bolysis. Browning et al. [57] describe an ultrasound system
which provides microbubble-mediated therapy to a thrombus
such as one causing ischemic stroke. Microbubbles are in-
fused or developed into the bloodstream in the vicinity of a
thrombus. Ultrasound energy is delivered to the microbub-
bles at the thrombus to disrupt or rupture the microbubbles.
This microbubble activity can in many instances aid in dis-
solving or breaking up the blood clot and return a nourishing
flow of blood to the brain and other organs. Such microbub-
ble activity can be used to deliver drugs encapsulated in mi-
crobubble shells, as well as microbubble-mediated
sonothrombolysis. Besides having enhanced thrombolytic
ability, combined use of ultrasound and microbubbles also
have a great potential for improved drug delivery across bio-
logic barriers. Kinoshita et al. [58] proved that microbubbles
combined with focused ultrasound improve the delivery of
trastuzumab a monoclonal antibody across the BBB.
2.3.4. Ultrasound Targeted Microbubble Destruction
(UTMD) for Chemotherapeutic Drug Delivery to Solid
Tumors
Cancer is a major cause of death worldwide. Current de-
livery methods of chemotherapeutic agent across the brain
are not very effective due to restriction provided by BBB.
4 Current Drug Targets, 2017, Vol. 18, No. 10
UTMD is a promising approach for the delivery of che-
motherapeutic drug to solid tumors [59, 60]. The encapsula-
tion of drugs in MBs reduces the harmful side effect of drugs
and provides subsequent local release at the targeted site by
ultrasound triggering. UTMD has been found effective in the
treatment of various malignant tumors like brain, liver, eye-
lid, pancreas, and breast [61-64]. In a preclinical study MB-
FUS in combination with liposomal doxorubicin provided
successful suppression of tumor [65]. Various studies have
been successfully evaluated the effectiveness of MB-FUS in
combination of chemotherapeutic agents in control of tumor
progression and in the improvement of the animal survival
[66-70].
2.3.5. Microbubble-facilitated Focused Ultrasound (MB-
FUS) in Gene Delivery
Ultrasound contrast agents composed of coated microbub-
bles have been widely studied as a means of gene delivery.
Microbubbles typically range between 0.5 to 8 µm in size and
have low solubility and diffusivity. Genetic material DNA,
RNA, siRNA, antisense oligonucleotides are usually intro-
duced into the microbubble and this protects the genetic mate-
rial from the immune system and also limits an unnecessary
immune response. Microbubbles are delivered intravenously
and can traverse unhindered through the smallest of blood
vessels resulting in an unparalleled ability to circulate
throughout the body vasculature and hence are ideal carriers of
genetic material for targeted delivery [71-74]. Ultrasound aids
both in enhancing cell permeability by creating small shock
waves and also aids in the release of the genetic material from
within the microbubble [75, 76]. Microbubble-facilitated fo-
cused ultrasound for brain targeted gene delivery was reported
in a study. This study evaluated the local, targeted delivery of
rAAV vector (recombinant adeno associated virus) for gene
therapy of CNS diseases, into the mice brains. Recently, FUS
mediated BBB opening in the presence of specially designed
plasmid conjugated microbubbles demonstrated the possibility
of local CNS gene expression [77].
2.4. Ultrasound Mediated Transcranial Nanoparticle De-
livery
Biodegradable polymeric nanoparticles with the smaller
size and desirable biocompatibility are a novel carrier for the
delivery of various kinds of drugs as they provide effective
drug protection, and target oriented delivery [78-80]. The
nanoparticles can be made sensitive towards pH, tempera-
ture, and light by including sensitive polymer chains and
chemical groups [81]. These attributes make nanoparticales
as a suitable mean for the delivery of drugs across the BBB.
So far the efficiency of microbubble incorporated polymeric
spheres and liposomes as ultrasound contrast agent, drug and
gene carrier has been proven. However, the immunogenicity
remains as a major problem for microspheres to clinical tri-
als. Therefore, the development of drug carriers that are
nanosized is very necessary [82, 83].
2.4.1. Focused Ultrasound/magnetic Nanoparticle Medi-
ated Drug Delivery
Magnetic nanoparticles (MNPs) have intrinsic magnetic
properties that enable their use as contrast agents in MRI
(magnetic resonance imaging) [84]. As the magnetic parti-
Nidhi Nainwal
cles are sensitive towards the external magnetic field, there-
fore magnetic targeting (MT) provide disposition of MNPs at
the target site. Focused ultrasound in the combination of
magnetic targeting synergistically delivers therapeutic MNPs
across the BBB [85]. A study combining FUS and MT of
nanoparticles confirmed that, on the concurrent application
of FUS and MT, BBB permeability in tumors increases [86,
87] (Fig. 2).
Transcranial magnetic resonance guided focused ultra-
sound (TcMRgFUS) was used in a study [88] on rats for the
delivery of Raman nanoparticles into brain tumors by re-
versible disruption of BBB. BBB disruption allowed the de-
livery of surface enhanced Raman scattering (SERS) capable
spherical gold nanoparticles (50 or 120 nm) to the tumor
region.
2.4.2. Ultrasound-triggered Thrombolysis Using Urokinase
Loaded Nanogels
In a study [89] polyethylene glycol cross linked glycol
chitoson hollow nanogels containing uPA (urokinase type
plasminogen activator) were formulated by ultrasonic spray
technique. The hollow nanogels with an inner cavity have
enhanced drug loading capacity. The nanogel shell gives
drug release in a programmable manner in response to envi-
ronmental pH and temperature. It was found that in compari-
son to nude urokinase, the uPA-loaded nanogels (200–300
nm) have a longer circulation lifetime, as the nanogels ma-
trix provide suitable protection and the thrombolysis rate of
blood clots also increases through the intervention of ultra-
sound [90, 91].
2.4.3. Ultrasound Mediated Sonothrombolysis by Nano-
droplet
It has been observed that the use of thrombolytics in the
form of nanosized system is more suitable for the manage-
ment of thrombolysis due to their ability to reach the throm-
bus through the narrow residual blood channels around the
arterial obstruction [92, 93]. In their study, Shimizu et al.
[94] reported preliminary results from an in vivo animal
safety evaluation in vivo experiment with superheated per-
fluorocarbon nanodroplets (SPNs). When triggered by ultra-
sound, these nanodroplets turn into microbubbles. During
this in vivo experiment, rabbits received either an IV injec-
tion of SPNs or a placebo without additional insonation. The
biochemical blood examination revealed no significant dif-
ferences between the SPN-treated group and the placebo
group. These researchers planned to conduct a study investi-
gating the SPN-assisted sonothrombolytic effect of 500-kHz
US exposure.
2.5. Stereotaxic Brain Atlas and Focused Ultrasound En-
ergy
The combination of stereotaxy with an anatomical atlas is
one approach for targeting specific regions in the brain. As
an alternative to MR-guided FUS, combined use of the
stereotaxic brain atlas and focused ultrasound energy allow
the efficient and economic studies (30 min per experiment
including preparation) in a conventional laboratory environ-
ment [95]. In one of the study carried out on rodent models
with a built-in rodent brain atlas, the application of the com-
Transcranial Focused Ultrasound (FUS) Triggered Brain Delivery
Fig. (2). (A) Intact CNS capillaries block delivery of MNP into the brain. (B) In the presence of microbubbles (MB), FUS temporarily dis-
rupts the BBB, enhancing passive influx of therapeutic MNPs at the target region. (C) Combining magnetic targeting with FUS actively tar-
gets the therapeutic MNPs to the brain. (A, astrocyte; EC, endothelial cell; N, neuron; P, pericyte).
pact stereotaxic-FUS system in transcranial localized BBB
opening is successfully verified [96].
2.6. Transcranial Color-coded Duplex Ultrasonography
(TCCS)
The visualization of basal cerebral arteries through the in-
tact skull by color-coding of blood flow velocity is enabled
by Transcranial color-coded duplex ultrasonography
(TCCS). In addition to the diagnosis of intracranial vascular
disease, TCCS is also helpful in intensive care and stroke
units, like follow-up examinations in vasospasm after suba-
rachnoid hemorrhage, and for intraoperative monitoring as
well. During the last two decades, TCCS found its important
role in the routine diagnostics of cerebrovascular diseases.
On the basis of advancement in computer and transducer
technology, TCCS emerged as a noninvasive method and has
a great scope in innovative imaging and therapeutic solutions
like cerebral perfusion imaging, sonothrombolysis, and site
targeted ultrasound contrast agents in brain delivery [97].
TCCS is found helpful in cerebral vascular malformations
such as arteriovenous malformation (AVM, angioma). With
TCCS, the pathological vascular convolutions of an AVM
can be displayed directly on the screen [98]. In a study [13]
venous cerebral echo color Doppler (ECD) ultrasound
evaluation is done in patients suffering from multiple sclero-
sis (MS). The result showed that chronic cerebrospinal ve-
nous insufficiency (CCSVI) syndrome is related to MS. A
significant correlation between these two diseases has al-
ready been outlined in previous studies. CCSVI seems to
hamper venous outflow from the brain towards the heart and
this might lead to a vascular congestion of the cerebral pa-
renchyma able to worsen the MS outcomes. Venous cerebral
echo-color Doppler ultrasound (ECD) evaluation can detect
venous morphological and hemodynamic parameter related
to CCSVI, and its results can be used to improve diagnostic
and therapeutic outcomes of patients suffering from MS [99-
102]. In another study, Ciciarello et al. [103] discussed the
age related vascular differences among patients suffering
from multiple sclerosis using echo color Doppler System.
2.7. Transcranial Magnetic Resonance-guided Focused
Ultrasound Surgery (TcMRgFUS)
For the non-invasive treatment of various brain diseases,
like uterine fibroids, painful bone metastases, adenomyosis,
Current Drug Targets, 2017, Vol. 18, No. 10 5
the tumor of breast etc. TcMRgFUS is an effective method.
Since MRgFUS is non-invasive, the risk of infection, blood
loss, and damage to nearby tissue is minimized [104]. In
general, magnetic resonance imaging (MRI) enables the ad-
justment of the US beam, based on differences in tempera-
ture measurements in the targeted parenchyma. As a primary
step, in vitro models using the human skull and porcine brain
have been developed for sonothrombolysis. In future, it may
be possible to detect the thrombus within the vessel, to focus
the US beam on this target, and make corrections to the US
beam so as to avoid side effects of US caused by distortion
and shifting of the human skull [105].
3. CONCLUSION
A novel system of non-invasive targeted BBB opening
by transcranial focused ultrasound is presented in this study.
The study shows that the FUS in conjunction with various
other techniques opens the BBB very effectively. The ad-
ministration of therapeutic agent using various FUS tech-
niques opens a new perspective in treating CNS diseases.
This review describes various methods and techniques in
favor of transcranial targeted brain drug delivery.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
Declared none.
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