Thrombosis Research (2008) 121, 799–811

intl.elsevierhealth.com/journals/thre

REGULAR ARTICLE

Improvement of in vitro thrombolysis employing

magnetically-guided microspheres
Michael D. Torno a , Michael D. Kaminski b,c , Yumei Xie a ,
Robert E. Meyers a , Carol J. Mertz b , Xianqiao Liu a ,
William D. O'Brien Jr. e , Axel J. Rosengart a,c,d,⁎

a
Neurocritical Care and Acute Stroke Section, Departments of Neurology, The University of Chicago Medical Center,
Chicago, IL, USA
b
Chemical Engineering Division, Argonne National Laboratory, Argonne, IL, USA
c
Collaborative Investigators for Applied Nanotechnology in Medicine, Department of
Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA
d
Surgery (Neurosurgery), The University of Chicago Medical Center, Chicago, IL, USA
e
Bioacoustics Research Laboratory, Department of Electrical and Computer Engineering,
University of Illinois at Urbana-Champaign, Urbana, IL, USA

Received 3 April 2007; received in revised form 3 August 2007; accepted 24 August 2007
Available online 17 October 2007

KEYWORDS Abstract
Thrombolysis;
Magnetic spheres; Significant shortcomings in clinical thrombolysis efficiencies and arterial recanaliza-
Ultrasound; tion rates still exist to date necessitating the development of additional
Tissue plasminogen thrombolysis-enhancing technologies. For example, to improve tPA-induced systemic
activator; clot lysis several supplementary treatment methods have been proposed, among
Magnetic field them ultrasound-enhanced tissue plasminogen activator (tPA) thrombolysis which has
already found some clinical applicability. The rationale of this study was to
investigate whether biodegradable, magnetic spheres can be a useful adjuvant to
currently existing tPA-induced thrombolysis and further enhance clot lysis results.
Based on an envisioned, novel thrombolysis technology – magnetically-guided, tPA-
loaded nanocarriers with triggered release of the shielded drug at an intravascular
target site – we evaluated the lysis efficiencies of magnetically-guided, non-
medicated magnetic spheres in various combinations with tPA and ultrasound. When
tPA was used in conjunction with magnetic spheres and a magnetic field, the lysis
efficiency under static, no-flow conditions improved by 1.7 and 2.7 fold for red and
white clots, respectively. In dynamic lysis studies, the addition of ultrasound and

⁎ Corresponding author. Neurocritical Care and Acute Stroke Section, Departments of Neurology and Surgery (Neurosurgery), The
University of Chicago Medical Center, 5841 South Maryland Avenue, MC 2030, Chicago, IL 60637, USA. Tel.: +1 773 702 2364; fax: +1 773
702 1469.
E-mail address: arosenga@neurology.bsd.uchicago.edu (A.J. Rosengart).

0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.thromres.2007.08.017
800 M.D. Torno et al.

magnetically-guided spheres to lytic tPA dosages resulted in both maximum clot lysis
efficiency and shortest reperfusion time corresponding to a 2-fold increase in lysis
and 7-fold reduction in recanalization time, respectively. Serial microscopic
evaluations on histochemical sections reconfirmed that tPA penetration into and
fragmentation of the clot increased with escalating exposure time to tPA and
magnetic spheres/field. These results delineate the effectiveness of magnetic
spheres as an adjuvant to tPA therapy accelerating in vitro lysis efficiencies beyond
values found for tPA with and without ultrasound. We demonstrated that the
supplementary use of magnetically-guided, non-medicated magnetic spheres
significantly enhances in vitro static and dynamic lysis of red and white blood clots.
© 2007 Elsevier Ltd. All rights reserved.

Introduction enhanced thrombolysis [56] as one recent clinical

Successful dissolution of intravascular blood clots
and cleavage of the fibrin network within the
thrombus depends on the joint effects of conversion
of activated plasminogen to plasmin as well as
effective exposure of both the substrate and the
plasminogen activator to the entire blood clot.
However, the delivery of plasminogen and activa-
tors, such as tissue plasminogen activator (tPA), into
the center of an occlusive vascular clot is frequently
limited as entry is either dependent merely on
passive diffusion [20], as seen in complete stasis
conditions, or reliant on pressure facilitated (bulk)
flow [48], which is not infrequently weakened by
inadequate collateral circulation or systemic hemo-
dynamic compromise. Furthermore, progression of
clot lysis with conventional thrombolytics proceeds
gradually and stepwise, allowing the thrombolytic
zone to only move slowly, layer-by-layer through the
clot, progressively restricting the amounts of tPA
available and, hence, lysis efficiency at deeper
thrombus sections [15,17].
To further improve tPA-induced clot lysis rates,
several supplementary treatment methods have been
proposed, among them ultrasound-enhanced throm-
bolysis which has already found some clinical
applicability [1,4,5,11,12,24,54]. Sonothrombolysis
was proposed more than one decade ago, when
ultrasound exposure of various intensities and dura-
tions were applied directly to blood clots in order to
accelerate clot lysis by increasing clot porosity and,
hence, the entry of surrounding plasma and thrombin
into the thrombus [15,21]. Preliminary clinical
experience indicates that the combination of sys-
temically-circulating tPA with adjunctive, external
ultrasound insonation may facilitate overall clot lysis,
shorten blood reperfusion time, and possibly lead to
an improved outcome in stroke patients [3,4].
Another example of supplementary thrombolytic
therapy is the systemic circulation of microbubbles
during lysis which may further accelerate ultrasound-
study found enhanced recanalization rates for other-
wise difficult-to-treat occlusions of skull-based ar-
teries [47]. Consisting of plain, echo-enhancing
galactose microspheres, originally designed as ultra-
sound contrast materials in heart imaging (i.e.,
Echovist), the spheres burst under ultrasound expo-
sure and, if within the clot vicinity, further enhance
clot porosity and breakdown [33,53].
The rationale for this study is the development of
an additional adjunctive method to further improve
clot lysis and recanalization rates. Our approach
investigates the use of magnetic designer nano- and
microspheres to mechanically enhance blood clot
porosity and to promote intraclot delivery of both the
thrombolytic and plasma. Our interest in testing such
magnetically-guided, bare magnetic spheres relates
to the initial building blocks of a new, targeted
thrombolysis treatment strategy that aims to improve
intravenous thrombolysis while reducing the risks
currently associated with catheter-based (intraarte-
rial) or high-dose (intravenous) tPA therapies [46]. In
this technology, magnetite (iron oxide crystals) is
encapsulated into biodegradable spheres made of
polymeric lactic acid shells [30,56]. Using external
magnetic fields, the nanocrystalline iron incorpora-
tion uniquely allows the spheres to be magnetically
guided and concentrated which can take place even
over distance (i.e., by magnets external to the body)
and independent of interfacing body structures (i.e.,
skull) making this a non-invasive, yet targeted
treatment approach. The co-encapsulating of tPA
into these magnetic spheres constitutes the synthesis
of a unique, magnetically- and ultrasound-responsive
tPA carrier allowing both shielded transport and
magnetic guidance of tPA even during prolonged
blood circulation as well as triggered drug release
under sonication [23,25,34]. We envision future
applications of this technology to employ the simple
intravenous injection of the medicated, magnetic
carrier which then systemically circulates but is
subsequently magnetically concentrated at the
Improvement of in vitro thrombolysis employing magnetically-guided microspheres
intravascular target. Simultaneous with the magnetic
concentration at the thrombus, triggered tPA burst
release from the carrier and on-demand thrombolysis
is induced by external ultrasound [56]. Hence, in
addition to direct release of tPA at the clot site, this
technology utilizes both the support of magnetic
force and sonication to penetrate and dissolve the
thrombus and the goal of this in vitro study was to
define whether bare magnetic spheres directed
magnetically into a blood clot will enhance clot lysis
in addition to the action of tPA and ultrasound.
The parameters investigated and described in
this study evaluate the lytic efficiencies of free tPA
in the presence or absence of either bare magnetic
spheres, ultrasound, or a combination of these
employing widely-accepted and frequently-
employed static and dynamic in vitro thrombolysis
models [24,52]. In the static flow experiments we
exposed standardized, radioactive-labeled blood
clots to various combinations of tPA and the test
materials, while the flow experiments simulate the
conditions present at common cerebrovascular
occlusion sites; i.e., arterial branch obliterations
[48]. In both models, the overall outcome was the
association between post-lysis clot reduction and
the utilized lysis variables. Furthermore, to better
simulate clinical thrombolysis scenarios, we studied
both red and white blood clots which occur in situ
under different pathological conditions and often
respond differently to thrombolytics [28]. Throm-
bolysis-adjuvant therapeutics such as ultrasound or
the here proposed magnet spheres may exert
additional lysis benefits in these settings [2,13–
16,20,21,25,46]. Finally, we compared immunohis-
tochemically the effects of tPA and magnetic
guidance on blood clot lysis using double-labeling
techniques and serial clot sectioning.
Materials and methods
Preparation and characterization of magnetic
nanosphases and magnetic spheres
To demonstrate and test our primary hypothesis that magneti-
cally-guided spheres are capable of increasing lysis efficacy by
means of clot penetration we synthesized spheres with appro-
priate magnetization values (that is, with proper generation of
magnetic drag forces once exposed to a permanent magnetic
field) rather than actual diameter in the nano-range.
Hydrophobic oleic acid-coated magnetite nanophases were
prepared by a modified co-precipitation method, as previously
described [30]. Poly(lactic acid)–poly(ethylene glycol) (PLA–PEG)
diblock copolymers were synthesized according to ring-opening
polymerization [43]. We purchased methoxy-PEG from Nektar
Company (San Carlos, CA, USA) and lactide monomer was kindly
supplied by PURAC Company (Lincolnshire, IL, USA). Molecular
weights (MW) of PLA–PEG were determined by permeation
chromatography (GPC, Bruker AM 400). Tetrahydrofuran (THF)
801
was used as the eluent and polystyrene as the standard. The
expected weight ratio of PLA to PEG was confirmed by 1H NMR
spectroscopy at 300 K using a Bruker Advance 500 spectrometer
operating at 500 MHz and with deuterochloroform as the solvent.
PLA–PEG magnetic microspheres were fabricated based on a
water-in-oil-in-water double emulsion technique. 300 mg of PLA–
PEG and 30 mg of oleic acid-coated magnetite (washed twice
before using) were dissolved in 3 mL of dichloromethane to form
the oil phase. The inner water phase containing 300 μL distilled
water was added to the oil phase and emulsified using an
ultrasound generator and sonication probe with a 4 mm tip size
(UltrasonicPower Corporation, Model 1000L Generator and 40TL
Transducer Probe, Freeport, Illinois) at an output power of 50 W
for 24 s. The resulting primary emulsion (water-in-oil emulsion,
W1/O) was injected drop-wise into the outer water phase
containing 90 mL of poly(vinyl alcohol) (PVA, 88% hydrolyzed,
MW = 22,000) (Acros Organics, New Jersey, USA) aqueous solution
(0.4 wt.%) under homogenization at 6000 rpm (Ultra Turrax® IKA
T18 Basic Homogenizer, IKA Works, Inc., North Carolina, USA) for
3 min in an ice bath. The resultant emulsion (water-in-oil-in-
water emulsion, W1/O/W2) was mixed at 200 rpm by a paddle
stirrer for 1 h to evaporate the dichloromethane solvent. To
ensure evaporation of the volatile solvent, nitrogen was bubbled
for an additional 1 h while stirring at 200 rpm. The magnetic
microspheres were isolated by a 4000 Gauss square magnet and
washed three times with 0.22 μm filtered deionized water. The
product was dispersed in 10 mL deionized water and frozen at
− 20 °C. An aliquot of the sample was lyophilized overnight to
determine the final microsphere concentration. The magnetiza-
tion of lyophilized magnetic microspheres, determined by a
vibrating sample magnetometer (VSM) (7400 Series, Lake Shore
Cryotronics, Inc Westville, Ohio, USA), was evaluated by sweeping
the applied field from 0 to 10 kOe and then to − 10 kOe at room
temperature. The morphology of the synthesized PLA–PEG
magnetic spheres was observed by scanning electron microscopy
(SEM, Quanta ESEM, FEI, Philips, 5.0 kV).
Ultrasound calibration
The same ultrasound generator and 4-mm tipped transducer-probe
used in the emulsification step described in the synthesis of
particles above was used in the thrombolytic experiments
described in this study. Ultrasound intensity of the transducer was
preliminarily calibrated at room temperature by UltrasonicPower
Corp (Freeport, IL) with a Clarke-Hess, Model 255 Watt meter
(Medford, NY) connected between the generator and transducer. A
70–80% power transfer efficiency is observed when calibrating with
the 4-mm US transducer/probe. Acoustic intensity remained a
constant variable (17.63 W/cm2) and was calculated by taking
power (50 W) over the surface area of the transducer or J = 50 W /
(πD2 / 4) where J is the acoustic intensity and D, diameter of
transducer tip (1.9 cm) [6,37]. According to company's power
calibration, only 70–80% of power is successfully transferred from
the US generator to the transducer. Thus, intensity (J) was
determined to be 13.2 ±1.2 W/cm2 given a 70–80% power transfer
efficiency.
Further calibration and measurement of acoustic pressure
generated by the ultrasound emitter was conducted at the
Bioacoustics Research Laboratory, Department of Electrical and
Computer Engineering at the University of Illinois Urbana-
Champaign. Briefly, a calibrated (52 mV/kPa at 20 kHz) omnidi-
rectional F42C model hydrophone (NUWC/USRD, Newport, RI)
was monitored by a digital oscilloscope (LeCroy 9354TM, Chestnut
Ridge, NY). The hydrophone was submerged in 2-L degassed H2O
at room temperature and positioned approximately 2 mm from
the US transducer that was inserted approximately 4 cm into the
802
water bath. Calibration of the hydrophone was based on
manufacturer's recommendation and on experimental conditions
in which the ultrasound transducer would be used for in vitro
thrombolysis described in this study [41,42].
A single pulse lasting 10 s, at 20 kHz and 50 W (13.2 ± 1.2 W/cm2)
yielded an acoustic pressure (zero-peak, p0) of 38–58 kPa. This
lower acoustic pressure was obtained due to visibly observable air
bubbles (cavitation) which manifested at the end tip of the
transducer and accordingly acted as a barrier between the probe
tip and hydrophone. To overcome the obstruction caused by
cavitation, the hydrophone was manually moved slowly and
horizontally along the surface of the transducer tip so as to clear
the air bubbles impeding the US from localized dispersion. Without
the occurrence of cavitation, an acoustic pressure of 135–154 kPa
was obtained, yielding an estimated probe displacement amplitude
at 20 kHz (p0 /ωρc) of 72–82 μm. In addition, using an acoustic
pressure of 140 kPa, its intensity is 6.5 W/cm2 which is within a
factor of 2 that was estimated from company information.
Blood clot preparation
Human blood was collected in K2EDTA Vacutainer tubes (Becton,
Dickinson and company, Franklin Lakes, NJ) and standardized red
and white blood clots were made according to a published
protocol [28]. In brief, whole blood was centrifuged at 130 g for
30 min and the platelet-enriched plasma (PRP) was transferred to
a separate tube. Cell-free plasma (CFP) was obtained by the
centrifugation of the blood for additional 10 min at 1400 g. The
platelet concentrations and hematocrit levels of PRP, CFP and
whole blood were determined by a Coulter® LH 750 hematology
analyzer (Beckman Coulter, Inc, Fullerton, CA, USA). Standard-
ized plasma was obtained by mixing PRP, CFP and whole blood to
form a consistent platelet concentration of 250 to 350 × 103/μL
and a hematocrit level of 0.5%.
White emboli were formed by maintaining a 5.3 μL to 1.3 μL
volume ratio of standardized plasma to thrombin solution
(0.75 U/μL) at room temperature in a 0.5 mL polypropylene
centrifuge tube. The final thrombin concentration in the clot was
Figure 1 Schematic diagram of dynamic flow model to evaluate pressure-driven thrombolysis under various test conditions.
This gravity-dependent flow system simulates a physiological volume rate of 2.1 mL/s such as seen in larger human arteries. At
time zero, different treatments such as tPA, MM, MF and US were introduced into the flow system. The reperfusion time as well
as the loss of clot radioactivity before and after treatment was measured to determine the lysis efficiency.
M.D. Torno et al.
maintained at 0.15 U/μL [28]. Red emboli were prepared by
mixing 2 μL whole blood per 0.5 μL thrombin solution (0.75 U/μL)
after the hematocrit level in the whole blood was adjusted to 30%
by the addition of PRP. Formation of the white or red emboli was
initially seen in 10–15 min after addition of thrombin, and stored
at room temperature for 24–48 h before use [28]. A five-second
centrifugation of the 0.5 mL tube proved to be useful in
supernatant extraction, before weighting the emboli. To produce
125
I-radiolabeled white and red blood clots, 125I-radiolabeled
fibrinogen was added to whole blood or standardized plasma to a
final 125I fibrinogen concentration of 0.4 μCi/mL [22]. The same
procedure was subsequently used to generate non-radiolabeled,
standardized white and red clots.
Thrombolysis models and study parameters
Standardization of lysis dose and time
Prior to testing the research variables and utilizing the thrombolysis
models we determined the optimal time and concentration required
to obtain maximal tPA lysis of the standardized red and white blood
clots. The literature describes effective tPA blood concentrations
leading to successful thrombolysis in humans to range from 1 to 4 μg/
mL [32,40]. However, larger tPA concentrations have also been
employed especially with local applications of the thrombolytic;
hence, we tested not only incremental tPA exposure times for up to
3 h but also a tPA concentration range from 0.5 to 15 μg/mL. Maximal
clot lysis, as determined by reduction of both clot weight and
radioactivity, was achieved with a local tPA concentration of 5 μg/
mL and an exposure duration of 45 min (data shown below);
therefore, these parameters were employed in subsequent throm-
bolysis experiments.
Test variables
Using bare PLA–PEG magnetic microspheres (MM) synthesized in
our laboratories, the standardized 125I radiolabeled red or white
blood clots were exposed in individual lysis experiments to one of
the following treatments: (1) magnetic microspheres (MM); (2)
MM plus magnetic field (MF); (3) MM plus 5 μg/mL tPA plus MF; (4)
Improvement of in vitro thrombolysis employing magnetically-guided microspheres 803

Figure 3 Clot lysis time versus lysis efficiency utilizing a free

tPA concentration of 5 μg/mL (red blood clots; n = 10, NS = non-
Figure 2 Scanning electron micrograph (SEM) of the synthe-
significant). The lysis efficiency in the 45 min exposure group
sized PLA–PEG magnetic spheres which were ∼20 μm in average
was maximal compared to the other test groups.
diameter.

ultrasound alone (US); and (5) tPA plus US; (6) MM plus tPA plus MF layer of liquid was carefully removed and the remaining clot
plus US. The treatment groups, the tPA concentration (5 μg/mL) weight was measured again by analytic balance. The respective
as well as the MM concentration (5 mg/mL) and MF strength lysis efficiency values were determined as the percentage
(Neodymium Iron Boron (NdFeB) magnet, 0.4 T at surface; Dexter decrease of the individual clot weight after treatment. For the
125
Industries) remained identical in all test series and for both the I-labeled clot, the pre-treatment radioactivity (Cobra™ auto-
static and dynamic lysis models. Further, the individual volumes gamma counter, Model 5002, Packard [PerkinElmer Life &
of tPA, MM and PBS as well as the temperature (37 °C) were kept Analytical Sciences, Shelton, CT, USA]) was determined by the
constant throughout the study to aid in accurate quantification of difference of radioactivity within the clot-containing tube, which
the results. The concentration of the bare MM was chosen to contained the mixed blood components and thrombin solution,
match the results of a previous reported mathematical analysis in and the supernatant of the aqueous solution after clot formation.
which the required 5 μg/mL tPA treatment dose can be released Post-treatment radioactivity of the remaining clot was counted
if the spheres were actually medicated with tPA [56]. Also, if after centrifugation of the tube and removal of the supernatant.
required by protocol, the magnetic field was applied for the first Lysis efficiency was expressed as the percentage radioactivity
20 min which then was immediately followed by a localized decrease after treatment.
single, 10 s, 6.5 W/cm2 ultrasound pulse at 20 kHz.
Immunohistochemical evaluation
Lysis models
All lysis experiments were performed at room temperature. In
the static lysis model the individual blood clot was placed at the We performed a series of immunohistochemical studies with
bottom of a tube which allowed the clot to occupy the complete fluorescently-labeled plasminogen and tPA to delineate the
inner tube circumference restricting the various treatments to
remain in contact only with the clot surface. The magnet was
placed below the clot, at the outer tube bottom. The dynamic
flow model aimed at duplicating some of the physio-anatomical
conditions related to a single artery embolic occlusion in humans
(Fig. 1). Individual blood clots were placed within a Tygon® lab
tubing (Cole Parmer Instrument Co., Vernon Hills, Illinois, USA) at
a reduction point where the inner diameter decreases from 3.8 to
3.1 mm. A fluid pressure gradient was formed using a gravity-
dependent flow system simulating a physiological volume rate of
2.1 mL/s such as present in larger human arteries. The flow was
started by the pressure gradient in the system. At time zero,
different treatments such as tPA, MM, MF and US were introduced
into the flow system. The reperfusion time for dissolution of the
clot among the different treatment groups as well as the
radioactivity loss of the clot before and after treatment was
measured to determine the lysis efficiency.
Figure 4 The effect of tPA concentration on clot lysis
Evaluation of lysis efficiency efficiency (red blood clots; lysis time 45 min; n = 10, NS =
non-significant). A tPA concentration of 5 μg/mL over 45 min
All non-radiolabeled clots were weighed on an analytic balance proofed optimal and also is in agreement with previously
before treatment. After treatment, the partially dissolved clots reported tPA concentrations resulting in clinically in effective
and the aqueous solutions were quickly centrifuged, the upper arterial thrombolysis.
804 M.D. Torno et al.

Figure 5 Static lysis. Lysis efficiency increased with the addition of each supplementary treatment method and was maximal
when tPA lysis was accelerated by the combined use of magnetic spheres/field and ultrasound (A). Among different blood clots
types, white clots showed improved lysis only for the tPA plus magnetic spheres/field groups (B; P b 0.04).

penetration depth of tPA into the clot and to demonstrate the
dissemination of magnetic spheres within the clot matrix. In brief,
sample clots from the above treatment groups were placed at 3
different time points (immediately, 1 min and 2 min after
treatment initiation) in an embedding container, fully covered
with OCT compound and cryostat frozen (HistoStat Microtome,
Reichert [Model 855, Buffalo, NY, USA]). The clot samples were
then cut in 4 μm thick slices using a plane parallel to the magnetic
field exposure, fixated in acetone and air dried. Antibody staining
was performed using primary antibodies (mouse IgG against human
plasminogen, dilution 1:50; rabbit IgG against human t-PA, dilution
1:100; dilutions) and secondary antibodies (goat anti-mouse IgG
and goat anti-rabbit IgG; Alexa 488 and 594, respectively; dilution
1:100) [14–16]. Digital imaging employing fluorescence-microsco-
py (Leica DM4000 B, Leica Microsystems Inc, Germany) was used for
semiquantitative analyses.
Statistical analyses
All data were expressed as mean ± standard deviation (SD). Two-
tailed Student's t-test for unpaired data was used to compare
means between two groups and for comparisons involving
multiple groups, one-way analysis of variance (ANOVA) was
Improvement of in vitro thrombolysis employing magnetically-guided microspheres 805

Figure 6 Lysis efficiency (dark columns; in %) and reperfusion time (blank columns; in seconds) employing a dynamic flow
system with a standardized red blood clot. Lysis efficiency increased significantly with the use of magnetic spheres/field alone
and further enhancement was seen with the use of ultrasound. However, the greatest lysis efficiency was noted for the tPA plus
magnetic spheres/field plus ultrasound group. The reperfusion times correlated inversely with the percentage of lysis efficiency
(ANOVA, P b 0.001 for all intergroup differences).

employed. Statistical significance was accepted at levels of
P b 0.05.
Results
PLA–PEG magnetic spheres
The SEM image (Fig. 2) of the synthesized PLA–PEG magnetic
spheres identified porous surface structures with a mean
diameter size of ∼ 20 μm. The specific saturation magnetization
of the synthesized spheres was ∼ 2.8 emu/g which is a relative
low value compared to most recent progress reported by our
group where values as high as 42 emu/g can be obtained [30].
Such significantly improved magnetization values greatly en-
hance our abilities to magnetically guide and concentrate the
spheres; however, as outlined in the immunohistochemical
analyses below, the present study utilized magnetic fields only
over short distances and avoided trapping of spheres against flow
gradients, hence, the obtained magnetization values were
sufficient to achieve complete magnetic clot penetration of
the spheres.
Thrombolysis time and tPA concentration
The time required for maximal tPA lysis efficiency was studied
utilizing red blood clots with an average, standardized clot weight of
71 ±4 mg and a tPA concentration of 5 μg/mL. The lysis efficiency
was 11.5±5.5%, 24.1±4.3%, 31.7±7.6% and 32.7 ±6.4% for free tPA
exposure times of 15, 30, 45 and 60 min, respectively (Fig. 3).
Student's t-test determined no significant difference among the 45
and 60 min exposure groups, while the efficiency in the 45 min
exposure group was maximal when compared to the 15 and 30 min
groups (Pb 0.0001, P b 0.015 respectively). Therefore, in all subse-
quent experiments a lysis time of 45 min was employed.
Using a tPA exposure time of 45 min and red blood clots we
tested three different tPA concentrations to determine the
optimal tPA concentration-to-lysis efficiency ratio for our
experimental set-up. We obtained lysis efficiencies of 17.0 ±
9.4%, 34.3 ± 3.3% and 41.4 ± 9.0 % for the respective tPA
concentrations of 0.5, 5 and 15 μg/mL (Fig. 4); only the lower
two concentration groups were significantly different (P b 0.001).
Therefore, we employed a tPA concentration of 5 μg/mL over
45 min exposure in all subsequent experiments; this value is in
agreement with previously reported tPA concentrations resulting
clinically in effective arterial thrombolysis [32,40].
Static thrombolysis
Comparing similar clot types, one-way ANOVA determined that
the various treatment groups were significantly different
(P b 0.0001; Fig. 5A). Lysis efficiency increased with the addition
of each supplementary treatment method. The lowest lysis
efficiency was found for bare magnetic microspheres (MM) used
as the sole treatment and resulted in 16 ± 6% and 15 ± 4% clot
reduction for the red and white clots, respectively. These results
did not change significantly with the addition of directed
magnetic fields (MF) to the spheres (MM + MF group; Fig. 5A).
However, lysis efficiency increased by 1.7 and 2.7 fold to 44 ± 7
and 62 ± 11 for red and white clots, respectively, when tPA was
added (MM + tPA + MF). The use of ultrasound (US) alone increased
the lysis efficiency to 39 ± 14% and 46 ± 11% for the red and white
clots, respectively, which was slightly less than for the MM + tPA +
MF group. However, when tPA lysis was accelerated by MM/MF
and US over 98% lysis efficiency was achieved for both white and
red clots (MM + tPA + MF + US group; Fig. 5A).
Comparing the lysis efficiency of the various test groups for
significant differences with respect to the clot composition
(Fig. 5B) we identified a significant difference only for the group
combining magnetic spheres/field with tPA: red clots 46 ± 5% and
806 M.D. Torno et al.

Figure 7 Immunofluorescent images of longitudinally-sectioned, untreated red blood clots identified (A) even distribution of
plasminogen (FITC; green) throughout the thrombus and no presence of tPA (Texas Red). (B) After exposure of the clots to magnetic
microspheres and fields only clot breakage from migrating spheres (inlet, ×100) is observed. (C) After treatment with tPA only
deposition of tPA is observed solely in the outer periphery with minimal penetration of the enzyme into the thrombus (composite). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

white clots 60 ± 9% (P b 0.02). All other test groups did not differ both greatest lysis efficiency and shortest reperfusion time which
significantly. correspond to an approximated 2-fold increase in lysis efficiency and
7-fold decline in recanalization compared to the control group
Dynamic lysis studies (magnetic spheres alone; 27±3% and 98± 2 s, respectively).

Pressure-dependent clot lysis, a commonly observed in vivo lysis Immunohistochemical studies

mechanism, was simulated with a simple dynamic flow system
employing flow occlusion induced by a red blood clot (Fig. 1). The
treatment groups and variable parameters were identical to
those utilized in the static lysis experiments; triplicates per test
group were obtained. The lysis efficiency (Fig. 6) enhanced
significantly (ANOVA, P b 0.001 for all intergroup differences)
with the use of magnetic spheres in the presence of a magnetic
field (36 ± 5%), and even further, when tPA or ultrasound was
added (44 ± 5% and 50 ± 4%, respectively). The greatest lysis
efficiency improvements, however, were noted for the combined
treatment with tPA, magnetic spheres/field and ultrasound (87 ±
3%). The time period to first reperfusion (Fig. 6) from 87 ± 3 s for
the use of magnetic spheres with magnetic field, 21 ± 2 s when tPA
was added, 15± 2 s with concomitant ultrasound delivery, and 11 ±1 s
for the combined use of tPA, magnetic spheres/field and ultrasound
(ANOVA, Pb 0.001 for all intergroup differences). Therefore, the
addition of ultrasound and magnetically-guided spheres resulted in
Immunohistochemistry and bright field microscopy were used to
provide semiquantitative estimates about the spatial distribution
of tPA, plasminogen and magnetic microspheres within red blood
clots after treatment exposure. Fig. 7 represents longitudinal
thrombus sections in which plasminogen appears green and tPA
red. Staining for plasminogen was evenly distributed throughout
the clots whereas no tPA staining was visible for untreated clots
as expected (Fig. 7A). Exposure of the clots to only magnetic
spheres and magnetic fields led to clot breakage caused by
penetrating spheres as identified on magnified views (Fig. 7B).
Lysis with tPA alone led to deposition of tPA solely at the top
layers of the thrombus identified on individual staining and
composite imaging (Fig. 7C). In contrast, the combination of tPA
with magnetically-driven microspheres (Fig. 8) delineated a
considerable increase in tPA clot penetration, predominantly
along the passage trajectories of magnetic sphere. Lysis front and
Improvement of in vitro thrombolysis employing magnetically-guided microspheres 807

Figure 8 Immunofluorescent images of longitudinally-sectioned red blood clots treated simultaneously with tPA and
magnetic spheres/field (double staining for plasminogen (FITC; green; on left) and tPA (Texas Red; middle) with composite
images on right). Cryostat fixation and serial sectioning were performed at three different exposure time points. (A) Immediate
fixation after treatment initiation (time zero) identified increased tPA intensity and hence penetration into the clot and along
the trajectories of the magnetically-driven spheres. This correlated with the early penetration of the spheres into the thrombus
as seen on microscopy (not shown). (B) One minute after treatment initiation further clot breakage and (C) at 2 min after
treatment almost complete clot lysis and degradation were observed. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

clot fragmentation increased with escalating exposure time as

seen on serial sections prepared immediately after (Fig. 8A) as
Discussion
well as 1 and 2 min post-tPA and magnetic sphere treatment
(Fig. 8B and C, respectively). On visual inspection of the sections,
the tPA concentration of tPA showed a marked increased in the The principal rationale of this study was to investigate
neighborhood of deposited magnetic spheres, along the clot whether biodegradable, magnetic spheres can be a
length and following the magnetic field gradients. useful adjuvant to currently existing plasminogen
808
activator-induced thrombolysis and further enhance
clot lysis results. Current limitations of intravascular
thrombolysis are thought to be primarily caused by the
limited penetration of systemically circulating plas-
minogen activators, such as tPA, into the core of the
thrombus. In vitro [16] and animal studies [5,28] as
well as investigations based on theoretical modeling
[8,17] identified that tPA delivery and hence the
magnitude of clot lysis is largely dependent on either
simple diffusion or bulk flow allowing, frequently, only
lysis of the upper-most clot layers [48]. These findings
are supported by clinical evidence identifying that
systemic thrombolysis can be a relatively ineffective
treatment option, for example, in the setting of larger
arterial occlusions at the base of the skull, such as the
carotid artery terminus, where only 10% of occlusions
are recanalized by IV tPA subjecting the great majority
of remaining patients to devastating ischemic strokes
[13]. This lysis failure is likely attributable to the
combination of two facts; first such proximal occlu-
sions are more than an order of magnitude larger than
that of more distal occlusions (i.e., 0.4 versus
0.03 mL, respectively) [56] therefore representing
an overwhelming clot burden for unsupported enzy-
matic digestion. Similar conclusions hold true for
acute coronary occlusions leading to myocardial
infarction [31]. Second, embolization is a common
mechanism for arterial occlusions such as strokes and
embolic clots originating from more central sources,
i.e., heart or aortic arch [47]. Frequently, those
clots had time prior to embolization to mature into
plasma-extruded, white blood clots or clots partially
calcified from enclosed atheromatous lesions repre-
senting difficult-to-lyse embolic materials [26,29,38].
To address this problem, ultrasound targeted at the
clot material during tPA lysis has recently been shown
to be a clinically useful, non-invasive, supplementary
thrombolysis method for stroke therapy [4,20].
Ultrasound leads to increased porosity and, depending
on the energy level employed, to the formation of
cavitations within the clot facilitating enhanced
passage of thrombolytics [8,21,27,50,54]. However,
clinical limitations of ultrasound-supported thrombo-
lysis (also named sonothrombolysis) are emerging. For
example, as the ultrasound energy emitted from the
device is not restricted to the clot itself, increased
heating, edema formation and possibly bleeding of
surrounding tissue have been found [11,12,45,49].
Based on an envisioned novel thrombolysis tech-
nology proposed and under development by our
group – magnetically-guided, tPA-loaded nanocar-
riers with triggered release of the shielded drug at
the target clot site [46] – we were interested
whether the magnetic guidance of non-medicated,
magnetic microspheres can further enhance tPA-
and ultrasound-mediated thrombolysis. Our hypoth-
M.D. Torno et al.
esis was that an externally applied magnetic field
linked with clot-surrounding magnetic spheres leads
to enhanced clot breakage due to magnetically
forced entry of spheres into the clot material
therefore improving clot lysis beyond the efficiency
currently observed for tPA plus ultrasound. In
support of this assumption are recent studies
reporting improved sonothrombolysis in the pres-
ence of freely-circulating ultrasound contrast
agents [10,19,36,53,56]. These small, air- or gas-
filled microbubbles are not only echoreflective but
also exhibit non-linear oscillations during insonation
resulting in bubble rupture and, when employed
with tPA during sonothrombolysis, can further
accelerate cavitations and fluid motion within the
thrombus [9,33,35]. A similar lysis-supporting mech-
anism can be proposed for magnetic nano- and
microcarriers which consist of biodegradable, poly-
meric spheres (Fig. 2). However, in contrast to ultra
sound microbubbles such spheres would provide the
additional advantages of shielded tPA transport to
the clot site [25] as well as magnetic guidance [30] to
and possibly into the thrombus.
To prove this hypothesis we exposed standard-
ized, radioactive blood clots of different composi-
tions to various combinations of tPA, which was first
optimized with respect to the required lytic
concentration and lysis time, and magnetic spheres
exposed to a magnetic field and ultrasound. Under
static, no-flow conditions lysis efficiency increased
with the addition of each supplementary treatment
method; lysis efficiency improved by 1.7 and 2.7
fold for red and white clots, respectively, when tPA
was used in conjunction with magnetic spheres and
a magnetic field. These values were similar to those
achieved by sonothrombolysis. However, when tPA
lysis was supported by both magnetic spheres/field
and ultrasound maximal lysis efficiency (∼ 98%) was
achieved for both white and red clots. Furthermore,
using the same experimental set-up, we found that
magnetic spheres/field-supported lysis significantly
favored the lysis of white blood clots, possibly
because white blood clots are cell-extruded, plate-
let-rich, dense fibrin meshes which are frequently
more difficult to lyse than red blood clots [28] and
the generation of magnetically-forced intraclot
channels may allow brisker substrate entry leading
to rapid clot disruption. Moreover, in the static
experiments no lysis efficiency, defined as reduc-
tion in residual clot radioactivity, was observed for
magnetic spheres/field alone; this is expected as
the mere presence of clot fragmentation induced by
the spheres in the absence of free tPA will not lead
to changes in overall clot volume.
The lysis efficiencies of the dynamic thrombolysis
experiments were on the average higher than those
Improvement of in vitro thrombolysis employing magnetically-guided microspheres
obtained from the static studies which can be
explained by the advantage of pressure-driven
bulk flow which enhances clot lysis up to 50-times
over diffusion-mediated, static lysis [7,8,17,55].
The most important findings of the flow studies
were that clot lysis increased significantly in all test
groups with the use of magnetic spheres/field. Most
notably, the addition of ultrasound and magnetical-
ly-guided spheres to lytic tPA dosages resulted in
both maximum clot lysis efficiency and shortest
reperfusion time corresponding to a 2-fold increase
in lysis and 7-fold reduction in recanalization time,
respectively (Fig. 6). This complements the results
from the static lysis experiments delineating the
effectiveness of bare magnetic spheres to not only
enhance lysis as a single, non-ultrasound-based
adjuvant to tPA therapy but also to maximize tPA
penetration and clot breakage beyond values seen
for tPA and ultrasound. In other words, magnetic
spheres/field and ultrasound are two adjuncts with
synergistic, supplementing effects on tPA lysis. Also,
the sole addition of magnetic spheres/field to the
thrombus in the dynamic flow experiments led to a
small increase in both lysis and reperfusion perfor-
mance. This can be explained by the fact that some
clot disintegration (and subsequent wash-out lead-
ing to reperfusion) occurred primarily from the
combination of hydrodynamic pressure and sphere-
induced clot channeling.
Our immunohistochemical studies defined that
the clots exposed to tPA alone showed tPA
penetration depth and lysis restricted solely to
the top layers of the thrombus (Fig. 7C). This has
been reported in previous studies and the limited
drug delivery into the thrombus is due to the
presence of tight fibrin meshwork [17,18,44]. In
contrast, the combination of tPA and magnetically-
driven microspheres delineated a substantial in-
crease in tPA clot penetration, predominantly
along the passage trajectories of the penetrating
magnetic spheres (Fig. 8). Furthermore, on micro-
scopic evaluation the lysis front and clot fragmen-
tation increased along the depth of the clot with
escalating exposure time of the substrates. Also,
tPA accumulation was visualized in areas where
magnetic spheres resided as deposits within the
clot (Fig. 8). These experiments provided direct
evidence that not only the exposure of the
thrombus to magnetic fields resulted in penetra-
tion of the spheres into and fragmentation within
the clot material but also that tPA co-localized with
the penetrating spheres explaining the improved
lysis efficiency results utilizing tPA and magnetic
spheres/field.
There are some limitations to our experiments.
The resulting clot lysis efficiency rates were ob-
809
tained under ‘ideal’ experimental conditions; that
is, employing the most favorable local tPA dosages
and exposure times as well as optimal insonation
and magnetic field alignments. Those conditions
are less frequently, if ever, met under in vivo lysis
situations and hence, our data provide only a
careful estimate of the results to be expected in
future studies in the living. Also, the proposed
magnetically-supported tPA-delivery technology
envisions the use of drug-loaded, magnetic spheres
with on-demand, ultrasound-triggered tPA burst
release. We do not know whether similar improve-
ments in clot lysis will be seen for tPA-loaded
magnetic carriers even though a theoretical model
utilizing cautious clinical and bioengineering
boundary conditions supports the concept that
similar local drug and sphere concentration can be
achieved under human physiological conditions
[56]. Lastly, we decided to employ only one
particular mode of ultrasound parameters to
simulate the effects of sonothrombolysis and it
can be argued that other frequencies and dosing
schemes may have different effects on clot lysis
efficiency. However, an in-depth review of thera-
peutic ultrasound parameters (such as intensity,
duration, mode, and frequency) utilized in pub-
lished in vitro and in vivo thrombolysis studies
identified a lack of studies defining the ultrasound
conditions which provoke maximal clot lysis under
both experimental and human conditions; further,
we found no consistency in the literature among the
different sonication parameters utilized (data not
shown). We based our decision on the here
employed ultrasound parameters on both the
published literature [5,39,51] as well as the fact
that our preliminary experiments (not shown here)
utilized these parameter settings to successfully
trigger release tPA from prototype, tPA-loaded
magnetic spheres synthesized in our laboratories
[25].
In summary, we demonstrated that the supple-
mentary use of magnetically-guided, non-medicat-
ed microspheres significantly enhances in vitro
static and dynamic lysis of red and white blood
clots. This finding is especially interesting with
respect to future novel technologies which may
utilize magnetically-guided, medicated (i.e., tPA-
loaded) magnetic spheres to treat and optimize the
lysis of in vivo intravascular occlusions.
Acknowledgements
This work is supported by The University of Chicago
Brain Research and the Cancer Research Founda-
tions, Chicago.
810
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