Online Appendix for the following JACC article
TITLE: A New Drug Delivery System for Intravenous Coronary Thrombolysis with
Thrombus Targeting and Stealth Activity Recoverable by Ultrasound
AUTHORS: Hiroyuki Kawata, MD, Yoshiko Uesugi, PhD, Tsunenari Soeda, MD,
Yasuhiro Takemoto, MD, Ji-Hee Sung, MD, Kiyotaka Umaki, PhD, Keiji Kato, PhD,
Kenichi Ogiwara, MD, Keiji Nogami, MD, Kenichi Ishigami, MD, Manabu Horii, MD,
Shiro Uemura, MD, Midori Shima, MD, Yasuhiko Tabata, PhD, Yoshihiko Saito, MD
APPENDIX
Online Methods
Preparation of the nanoparticles encapsulating tPA
Briefly, basic gelatin (100,000 MW, Nitta Gelatin Inc., Osaka, Japan) and tPA
(monteplase; Cleactor® Injection, Eisai Co., Ltd., Tokyo, Japan) were mixed at the
concentration of 20 mg/ml and 1.0 mg/ml in 10 mM phosphate-buffered solution (PBS,
pH 7.4), respectively. After agitation, zinc acetate (Nacalai Tesque Inc., Kyoto, Japan)
was added to the mixture of basic gelatin and tPA (at final concentration of 5 mM) to
stabilize and tighten their connection (1,2) (Figure 1A). The diameter of the
nanoparticles is approximately 100 nm and its activity was reduced to approximately
50 % of unmixed tPA and recovered by low frequency US (3) (Supplemental Figure 4).
Measurement of the molecular size of nanoparticles
To measure the apparent molecular size of the nanoparticles, dynamic light scattering
(DLS) measurements were carried out using a DLS-7000 instrument (Otsuka
Electronics, Osaka, Japan) equipped with an Ar+ laser at a detection angle of 90° at
37°C (3,4). Three measurements were independently performed for each sample.
Assay of tPA activity in vitro
To determine tPA activity in vitro, a fibrin clot lysis assay was performed on the basis of
the plasminogen-rich fibrin plate method as previously described (4,5). Plasmin,
plasminogen, and thrombin isolated from human plasma were purchased from EMD
Biosciences Inc., California, USA. Briefly, 10 ml of 0.4 wt% human fibrinogen
solution in 0.17 M borate buffer (pH 7.8) was poured into a flat 10 cm Petri dish.
Plasminogen (0.2 ml, 50 U/ml) was added to the fibrinogen solution. After adding 0.2
ml of human thrombin (100 U/ml) to induce clot formation, the mixture was left at
room temperature for 30 minutes to allow for clot formation. Then, 2.0 l of tPA or
nanoparticles before or after 5 minutes of US application (continuous wave, 1.0 MHz in
frequency and 1.0 W/cm2 in intensity) in vitro were placed in PBS onto the plate.
After incubation at 37°C for 1 hr, the diameter of clear zones was measured while the
standard curve between the diameter and the activity was prepared using human plasmin
at determined concentrations. The percent activity was calculated as the percentage of
sample activity to that of tPA alone. The experiment was independently performed in
triplicate for each sample.
Animal experiments
The experiments in mice and rabbits were carried out according to the Institutional
Guidance on Animal Experimentation of Kyoto University and Nara Medical University,
respectively. The experiments in swine were performed at the laboratory of the
Interventional Technical Center (IVTeC Co., Ltd., Kobe, Japan) and were approved by
the Animal Experimental Committee of the IVTeC.
Measurement of plasma tPA activity in rabbit and swine models
To evaluate whether tPA activity was suppressed by nanoparticles in vivo, in rabbits,
blood samples from the femoral artery (FA) were collected just distal to the site of
transcutaneous US application immediately, 5, 15, 30, 45, and 60 minutes after the
injection of tPA or nanoparticles (n=4 for each treatment). In the swine experiments,
blood samples from the left coronary artery (LCA) and right FA were collected
separately in each swine immediately (before the US application in the swine treated
with transthoracic US), 10, 15, 30, 45, and 60 minutes after the injection of tPA or
nanoparticles (n=5 for each treatment).
The plasma tPA activity of each blood sample was measured with the synthetic
substrate, chromozym tPA, according to the manufacturer’s instructions (Roche Applied
Science, Indiana, USA).
Data were expressed as the g/ml equivalent of control tPA.
In vitro vWF binding assay
To evaluate whether vWF protein binds to tPA, basic gelatin, or nanoparticles, enzyme
immunoassay (EIA) was performed with the use of rabbit anti-human vWF antibody
(DAKO, Glostrup, Denmark) as described previously (6,7) with minor modifications.
A 96-well plate was coated with 100 l of anti-human vWF antibody (1.0×10−8 M) as a
control. The experimental wells with tPA, basic gelatin, or nanoparticles were coated
with 100 l of tPA (1.0×10−6 M), basic gelatin (1.0×10−6 M), or nanoparticles
containing basic gelatin (1.0×10−6 M). Subsequently, 50 l of vWF protein (8.0×10−7
M) was added to each well. Horseradish peroxidase (HRP)-conjugated anti-human
vWF antibody (DAKO, Glostrup, Denmark) was added to each well. The absorbance
of each reaction was measured at the wavelength of 492 nm.
Preparation of the mouse arterial thrombosis model
Thrombotic occlusion of the left FA in 10 female ddY mice aged 6 weeks was induced
by ferric chloride methods as previously described (8,9). The left FA of each mouse
was exposed and wrapped with a 5 mm-wide strip of filter paper saturated with 30 %
FeCl3 to injure the vessel wall and induce thrombotic occlusion.
Evaluation of tPA accumulation in the occluded vessel
For the evaluation of tPA accumulation at the thrombus site in the mouse arterial
thrombosis model, tPA was radioiodinated with 125I according to the chloramine T
method as previously described (10).
After iodination, tPA activity was not reduced, suggesting that iodination procedure
itself did not affect tPA activity.
125
I-labeled tPA alone (27,500 IU/kg; 0.2235 mg/kg)
or nanoparticles containing the same dose of 125I-labeled tPA (n=5 for each) in 100 l of
PBS were injected into the tail vein of the mouse 30 minutes after the induction of
thrombotic occlusion in the left FA. Ten minutes later, the bilateral iliofemoral arteries
at 2 cm length were excised along with the blood inside the lumen after ligation of both
ends and all branches. The radioactivity of the iliofemoral artery was counted with a
gamma counter (ARC-301B, Aloka, Tokyo, Japan). The radioactivity of the affected
left iliofemoral artery was expressed as a ratio of the radioactivity in the contralateral
normal iliofemoral artery.
Preparation of the rabbit arterial thrombosis model
For the preparation of the rabbit arterial thrombosis model, balloon injury of the right
femoral artery (FA) was induced according to the procedure reported previously, with
modifications (11-13). Twenty male Japanese white rabbits (2.5–2.7 kg; SLC Japan,
INC. Shizuoka, Japan) were anesthetized with pentobarbital sodium (25 mg/kg, iv).
The first injury was induced by fluoroscopically inserting a PTCA balloon catheter
(diameter, 3.0 mm; length, 8.0 mm) via the left carotid artery to a distal site in the right
FA where it was inflated to 8 atm. The inflated balloon was then pulled back a
distance of 3 cm thrice. After the formation of a stenotic lesion was confirmed
angiographically 4 weeks later, a second injury was induced. Another PTCA balloon
catheter (diameter, 2.75 mm; length, 15 mm) was inserted via the right carotid artery to
a site just distal to the stenotic lesion, and the second injury was induced in the same
manner as the first. Thereafter, blood flow was reduced to approximately 10% by
incomplete ligation distal to the injured area, which caused immediate formation of an
occlusive thrombus. To confirm the presence of a thrombotic occlusion, angiography
was carried out using a catheter inserted into the abdominal aorta 15 minutes and 2
hours after the second injury.
Thrombolysis in the rabbit model
Immediately after confirmation of right FA occlusion, thrombolysis was initiated by the
injection of 27,500 IU/kg (0.2235 mg/kg) of tPA, which corresponds to the clinical dose
used in humans, or nanoparticles containing the same dose of tPA via an ear vein. In
cases treated with US, continuous wave US (1.0 MHz, 1.0 W/cm2) was simultaneously
applied transcutaneously over the thrombus up to 60 minutes until successful
thrombolysis was obtained (n=10 in each treatment). The ultrasound device comprised
of a cylindrical probe, 3 cm in diameter and 6 cm in length, connected to a power
amplifier. The effects of thrombolysis were evaluated angiographically.
Angiography was carried out every 15 minutes up to 60 minutes after the initiation of
thrombolysis, and the Thrombolysis in Myocardial Infarction (TIMI) flow grade was
determined.
Generation and evaluation of the transthoracic ultrasound device
Based on the in vitro and rabbit experiments, we tested US probes of 3 different
frequencies (0.5, 1.0, and 2.0 MHz) and 1.0 W/cm2 in intensity. Four US probes of
each frequency were combined in parallel as shown in Supplemental Figure 1 to
generate continuous wave transthoracic US (TUS) devices for the swine model. Only
the 1.0 MHz frequency (continuous wave, 1.0 W/cm2) device produced relatively
uniform US fields in a water bath test, as quantified by an oscilloscope. In contrast,
TUS with the 0.5 or 2.0 MHz devices produced a protrusive US field at each probe
(data not shown). In addition, the 1.0 MHz TUS device created uniform US fields
over the swine heart as quantified by a catheter-type oscilloscope inserted into the left
ventricle (LV), right coronary artery (RCA), left anterior descending coronary artery
(LAD), and left circumflex coronary artery (LCx) as shown in Supplemental Figure 2.
Preparation of the swine AMI model
For the preparation of the swine AMI model, thrombotic occlusion of the LCx was
induced in 60 female swine (35–45 kg) by balloon injury with distal balloon occlusion
as shown in Supplemental Figure 3. A 0.014 inch guide wire with a 4 mm balloon at
the tip was inserted into the mid-portion of the LCx. The balloon was then inflated to
occlude the LCx. The PTCA balloon (diameter 3.5 mm; length 10 mm) was inserted
over the guide wire to just proximal of the occlusion balloon, and inflated to 5 atm.
The inflated PTCA balloon was then pulled back a distance of 3 cm thrice. After 30
minutes with distal balloon occlusion, thrombotic occlusion of the LCx was confirmed
by coronary angiography (CAG). After an additional 30 minutes without distal
occlusion, the occlusion of the LCx was reconfirmed.
To demonstrate that the thrombus in the swine model was rich in platelets like that of
AMI in humans, parts of the thrombi were extracted, fixed in 4 % paraformaldehyde,
embedded in paraffin, and cut into 3 m sections, and immunostaining for GP IIb/IIIa
(Affinity Biologicals Inc., Ontario, Canada) was performed.
Thrombolysis in the swine AMI model
After the reconfirmation of occlusion of the LCx, thrombolysis was initiated by the
injection of 27,500 IU/kg (0.2235 mg/kg) or 55,000 IU/kg (0.447 mg/kg) of tPA, or
nanoparticles containing the same doses of tPA via an ear vein over 5 minutes.
Subsequently, continuous wave US (1.0 MHz, 1.0 W/cm2) was applied transthoracically
in the cases with TUS for up to 60 minutes (n=10 for each treatment). The position of
the TUS device was fluoroscopically confirmed to be located over the heart as shown in
Supplemental Figure 1B.
Angiography was carried out every 15 minutes up to 60 minutes after initiation of
thrombolysis treatment, and the TIMI flow grade was determined.
Evaluation of LVEF after thrombolysis
At 60 minutes after the initiation of thrombolysis, left ventriculography at left anterior
oblique view was carried out to calculate LVEF.
Gene expression of apoptotic or inflammatory markers
To evaluate the effects of US (continuous wave, 1.0 MHz and 1.0 W/cm2) on the gene
expression of apoptotic or inflammatory markers, 4 mice (C57BL/6, CLEA Japan, Inc.
Tokyo, Japan) were exposed to transcutaneous and transthoracic US for 60 minutes.
The hearts, lungs, livers, and kidneys were harvested from these experimental animals
as well as 4 control mice without US exposure, and RNA was isolated from each tissue
sample. The expression of bax, caspase 3, and IL-6 mRNA were evaluated by
real-time polymerase chain reaction (PCR).
Online Table 1.
Angiographic parameters (60min)
tPA
TIMI grade
mean TIMI grade
DDS
0 : n=3
(30%)
1 : n=2
0 : n=8
(80%)
1 : n=2
0 : n=0 (0%)
1 : n=0 (0%)
2 : n=0 (0%)
(20%)
2 : n=4
(40%)
3 : n=1
(10%)
(20%)
2 : n=0 (0%)
3 : n=0 (0%)
3 : n=10
(100%)
1.30 ± 1.06
††p<0.01 vs NP, **p<0.01 vs
NP
NP
††
0.20 ± 0.42
3.00
**
**
Online Figure 1.
(A) Illustration of the transthoracic ultrasound (US) device consisting of 4 probes.
(B) The left photo shows transthoracic US application in swine. The right
fluoroscopic image shows the positioning of the transthoracic US device.
Online Figure 2.
The US fields in the swine heart as quantified by a catheter-type oscilloscope
inserted into the indicated structure under transthoracic US application
(continuous wave, 1.0 MHz, and 1.0 W/cm2).
Online Figure 3.
(A) Coronary angiographic image showing distal balloon occlusion (arrow) for the
induction of thrombosis in the left circumflex artery (LCx).
(B) Coronary angiographic image showing thrombotic occlusion (arrowhead) in the
LCx 30 minutes after distal balloon occlusion.
(C) Thrombi obtained from the occluded LCx were strongly immunopositive for GP
IIb/IIIa.
Online Figure 4.
tPA activity of the nanoparticles (NP) before and after 5-minute ultrasound (US)
application expressed as the percentage of control tPA (*p<0.05).
20
plasm
a tPA
activi
ty
tPA
NP+late US
*
1
5
10
(g/m
l
5
equiv
alent)
0
*
*
0
5
15
†
†
*
30
min
45
6
0
Online Figure 5.
Time course of plasma tPA activity expressed as the g/ml equivalent of control
tPA in rabbit injected with 27,500 IU/kg of tPA or nanoparticles (NP)
intravenously. Transcutaneous US application was initiated 40 minutes after NP
injection in the cases of NP+late US. Blue arrow indicates the duration of
transcutaneous US application in NP+late US.
vs tPA at each time points.
*p<0.01 vs NP+late US, †p<0.01
Online Figure 6.
(A) Typical angiographic images at 60 minutes in rabbits treated with tPA (27,500
IU/kg) alone, nanoparticles (NP) alone, and the drug delivery system (DDS).
Arrowheads indicate the proximal site of thrombotic occlusion before treatment.
(B) Time course of thrombolysis after injection of tPA (27,500 IU/kg) or nanoparticles
(NP) in rabbits treated with tPA alone (▲), NP alone (●), and DDS (■) (*p<0.05
and **p<0.01 vs. tPA alone or NP alone).
Online Figure 7.
Gene expression of bax, caspase 3, and IL-6 in the heart (A), lung (B), liver (C),
and kidney (D) with transcutaneous or transthoracic ultrasound (US) application
(white column) or without (black column). Induction of each gene was not
observed with US application.
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Online Video 1.
A typical coronary angiography at 60 minutes in swine treated with tPA alone
(55,000 IU/kg).
Online Video 2.
A typical coronary angiography at 60 minutes in swine treated with tPA (55,000
IU/kg) followed by transthoracic US application.
Online Video 3.
A typical coronary angiography at 60 minutes in swine treated with the intelligent
drug delivery system (DDS).