Development of double-stranded siRNA labeling method using positron emitter and its in vivo trafficking analyzed by positron emission tomography

Article abstract of DOI:10.1021/bc9005267

Pharmacokinetic study of small interfering RNA (siRNA) is an important issue for the development of siRNAs for use as a medicine. For this purpose, a novel and favorable positron emitter-labeled siRNA was prepared by amino group-modification using N-succi

Full text of DOI:10.1021/bc9005267

756
Bioconjugate Chem. 2010, 21, 756–763
Development of Double-Stranded siRNA Labeling Method Using Positron
Emitter and Its In Vivo Trafficking Analyzed by Positron Emission
Tomography
†
†
†
†
†
‡
Kentaro Hatanaka, Tomohiro Asai, Hiroyuki Koide, Eriya Kenjo, Takuma Tsuzuku, Norihiro Harada,
‡
,†
Hideo Tsukada, and Naoto Oku*
Department of Medical Biochemistry and Global COE Program, Graduate School of Pharmaceutical Sciences, University of
Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-city, Shizuoka 422-8526, Japan, and PET Center, Central Research Laboratory,
Hamamatsu Photonics K.K., 5000 Hirakuchi, Hamakita-ku, Hamamatsu-city, Shizuoka 434-8601, Japan. Received
December 7, 2009; Revised Manuscript Received February 8, 2010
Pharmacokinetic study of small interfering RNA (siRNA) is an important issue for the development of siRNAs
for use as a medicine. For this purpose, a novel and favorable positron emitter-labeled siRNA was prepared by
18
amino group-modification using N-succinimidyl 4-[fluorine-18] fluorobenzoate ([ F]SFB), and real-time analysis
18
of siRNA trafficking was performed by using positron emission tomography (PET). Naked [ F]-labeled siRNA
1
8
or cationic liposome/[ F]-labeled siRNA complexes were administered to mice, and differential biodistribution
of the label was imaged by PET. The former was cleared quite rapidly from the bloodstream and excreted from
the kidneys; but in contrast, the latter tended to accumulate in the lungs. We also confirmed the biodistribution
of fluorescence-labeled naked siRNA and cationic liposome/siRNA complexes by use of a near-infrared fluorescence
imaging system. As a result, a similar biodistribution was observed, although quantitative data were obtained
only by planar positron imaging system (PPIS) analysis but not by fluorescence in vivo imaging. Our results
indicate that PET imaging of siRNA provides important information for the development of siRNA medicines.
INTRODUCTION
To evaluate the in vivo behavior of siRNA itself, the trafficking
of it is carried out by near-infrared fluorescence (NIRF) imaging
Small interfering RNA (siRNA) is a short double-stranded
nucleic acid molecule that induces sequence-dependent gene
silencing (1, 2), and gene therapy using siRNA is expected to
be a novel treatment strategy for various diseases (3). However,
the degradation of naked siRNA after administration into the
bloodstream of human and animals readily occurs due to
nucleases in the blood. Furthermore, siRNA poorly penetrates
the plasma membranes of target cells, thus making difficult the
delivery to the cytosol of targeted cells for effective gene
knockdown. Therefore, a delivery system of siRNA molecules
is considered to be indispensable for establishing siRNA therapy.
Many studies on the in vivo application of siRNA using
chemical modification (4) or drug delivery system (DDS)
carriers such as liposomes (5) and micelles (6) for efficacious
delivery and gene silencing have been reported. Cationic
liposomes or micelles are representative carriers having electric
charges on their surface. Because siRNA possesses polyanionic
charges, it electrostatically interacts with cationic carriers used
for RNA interference (RNAi). Pharmacokinetic studies on
siRNA/carrier complexes have been performed by using fluo-
rescence- or radioisotope-labeled carriers or carrier-entrapped
imaging agents for magnetic resonance imaging. The techniques
of carrier-labeling, however, do not always reflect the biodis-
tribution of siRNA, since there is a chance that the siRNA may
become detached from the carrier in the bloodstream, especially
when the siRNA is bound to the surface of cationic carriers.
(
7). However, like fluorescence imaging, NIRF imaging is
affected by tissue depth to some extent, and therefore, quantita-
tive data are difficult to obtain. In addition, although the NIRF
imaging method is applicable for use on nonhuman primates,
the method for human use has not yet been established.
To obtain precise pharmacokinetic information on siRNA
molecules or their carriers in vivo, positron emission tomography
(
PET) is one of the ideal techniques. PET enables the deter-
mination of the real-time biodistribution and topical accumula-
tion of positron emitter-labeled compound noninvasively. This
technique can be applied in preclinical studies, in which a drug
candidate labeled with a positron emitter is injected into animals.
The circulation profile, biodistribution in various tissues, and
eventual elimination of the drug candidate from the body can
be monitored noninvasively in the same animal. Furthermore,
certain positron emitter-labeled new drug candidates can be used
for microdosing studies in humans. Such phase zero studies
enable the investigator to reduce the rate of dropout of drug
candidates in further clinical studies. For the pharmacokinetic
1
8
study of DDS carriers, we previously reported a novel [ F]-
probe for labeling the lipid assembly carriers for PET analysis
(
8). As mentioned above, since siRNA may possibly dissociate
from its carrier and be degraded by plasma nucleases after
injection into the bloodstream, we have sought to label siRNA
with a positron emitter for the analysis of siRNA trafficking in
vivo. The pharmacokinetic information on siRNA molecules,
like that of the carrier, is considered to be critical and
indispensable for the development of siRNA medicines.
Recently, Bartlett et al. labeled the sense strand of siRNA
with copper-64 by using 1,4,7,10-tetraazacyclododecane-
N,N′,N′,N′′′-tetraacetic acid (DOTA) and then annealed this
modified strand with the unmodified antisense strand for the
determining biodistribution of siRNA in micellar carriers (9).
*
Corresponding author. Naoto Oku, Department of Medical Bio-
chemistry, University of Shizuoka School of Pharmaceutical Sciences,
5
5
2-1 Yada, Suruga-ku, Shizuoka 422-8526, Telephone number: +81-
4-264-5701, Fax number: +81-54-264-5705, E-mail address: oku@
u-shizuoka-ken.ac.jp.
†
University of Shizuoka.
‡
Hamamatsu Photonics K.K.
1
0.1021/bc9005267  2010 American Chemical Society
Published on Web 03/08/2010

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8
Synthesis and PET Imaging of [ F]-Labeled siRNA
Bioconjugate Chem., Vol. 21, No. 4, 2010 757
Viel et al. reported the conjugation of 18F to oligonucleotide by
system (Fuji Film, Tokyo, Japan), respectively. Fluorescence
imaging was performed by using a Xenogen IVIS Lumina
System coupled to LiVing Image software for data acquisition
(Xenogen Corp., Alameda, CA). PET imaging was performed
by using a planar positron imaging system (PPIS, Hamamatsu
Photonics, Shizuoka, Japan). Radioactivities in each organs were
measured by gamma-counter (ARC-2000, Aloka, Tokyo, Japan).
Experimental Animals. Five-week-old male BALB/c mice
male were purchased from Japan SLC Inc. (Shizuoka, Japan).
The animals were cared for according to the Animal Facility
Guidelines of the University of Shizuoka. All animal experi-
ments were approved by the Animal and Ethics Review
Committee of the University of Shizuoka.
18
using (N-[3-(2-[ F]fluoropyridin-3-yloxy)-propyl]-2-bromoac-
18
etamide ([ F]FRyBrA), and this method also required the
annealing process (10, 11). In spite of their efforts, detail
pharmacokinetic information about naked siRNA or siRNA in
DDS carriers is still largely lacking.
In the present study, we developed a novel technique for
labeling siRNA with a positron emitter, 1 F, in which double-
stranded siRNA was labeled to gain conformational accuracy
8
18
for examining the pharmacokinetics of siRNA by using [ F]SFB
as an 18F labeling reagent. [ F]SFB have been widely used to
form a stable amide bond by reacting with primary amino groups
for a short time and very easily for labeling certain peptides
18
(
12), antibodies (13), or DNA oligonucleotides (14). Because
F has an extremely short half-life (109 min), avoidance of
siRNA Sequence. Alexa Fluor 750-labeled siRNA (AF750-
siRNA) was purchased from Japan Bio Services Co., Ltd.
1
8
the annealing process by labeling of double-stranded siRNA is
(
Saitama, Japan). Antisense strand contained a fluorescence at
18
favorable. [ F]-labeled siRNA thus prepared was identified by
ESI-TOF-MS, HPLC, and autoradiography after electrophoresis.
By use of this positron emitter-labeled siRNA, we actually
examined the biodistribution of siRNA by PPIS in the presence
or absence of cationic liposome, a DDS carrier of siRNA. We
also performed NIRF imaging for verifying the in vivo behavior
of siRNA and discussed the correspondence of the imaging
results obtained from NIRF imaging and PPIS imaging.
the 3′ end of the strand. siRNA for positron emitter-labeling
was synthesized and purified by Hokkaido System Science.
The nucleotide sequences of the siRNA were those of an
unspecific scrambled RNA, and the antisense strand contained
a 3′ amino C6 linker for the radioactive labeling of the siRNA
duplex (Scheme 1). The sequences of the siRNA used were
5
′-CGAUUCGCUAGACCGGCUUCAUUGCAG-3′ (sense) and
5
′-GCAAUGAAGCCGGUCUAGCGAAUCGAU-3′ (antisense).
Synthesis of Ethyl-(4-Trimethylammonium)benzoate Tri-
fluoromethanesulfonate. Ethyl 4-dimethylaminobenzoate was
EXPERIMENTAL PROCEDURES
synthesized according to the procedure reported by Haka et al.
Materials. 4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]-
1
(
15). The synthetic compound was assigned by H NMR and
C NMR. Ethyl 4-dimethylaminobenzoate 11.4 g (59 mmol)
hexacosane (K[2.2.2]), tetra-n-propyl-ammonium hydroxide (Pr
N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluorobo-
rate (TSTU), CH CN (anhydrous), and fetal bovine serum were
obtained from Sigma-Aldrich (Saint Louis, MO, USA). Potas-
sium carbonate ·1.5H O was purchased from Merck (Darmstadt,
4
NOH),
1
3
was dissolved in 70 mL anhydrous CH
CF SO CH (64.9 mmol) was added dropwise to the solution,
and the mixture was stirred overnight at room temperature. A
crystalline precipitate was formed by the addition of Et O (100
mL) and collected by suction filtration. Recrystallization from
CH Cl /Et O gave 10.2 g (48%) of ethyl 4-trimethylammoni-
umbenzoate trifluoromethanesulfonate.
2 2
Cl . Ten grams of
3
3
3
3
2
-
2
Germany). Anion-exchange resin AG1-X8 (OH form, 100-200
mesh) was from Bio-Rad Laboratories (Hercules, USA). N,N-
Dimethylformamide (DMF) was purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). A cationic lipid for
transgene use, 1,2-dioleoyl-3-trimethylammonium-propane (DOT-
AP), was purchased from Avanti Polar Lipids Inc. (Alabaster,
AL, USA). Cholesterol was kindly provided by Nippon Fine
Chemical Co., Ltd. (Takasago, Hyogo, Japan). Trizol reagent
was obtained from Invitrogen Co. (Carlsbad, CA). An alfalfa-
free feed was purchased from Oriental Yeast co. Ltd. (Tokyo,
Japan). Escain was purchased from Mylan Pharmaceuticals
2
2
2
18
18
Radiosynthesis of [ F]KF/K[2,2,2] Complex. [ F]KF/K-
[
(
2,2,2] was obtained by use of a previously reported method
18
16). In brief, [ F]fluoride was trapped by ion-exchange resin
CO
CN (2 mL)
was added. Water was removed by azeotropic distillation at 110
C under He flow (400 mL/min) for 5 min. To the residue, the
addition of CH CN (1 mL) and azeotropic distillation were
AG1-X8 and eluted from the resin by 0.5 mL of 40 mM K
To this fluoride solution, 15 mg of K[2,2,2] in CH
2
3
.
3
°
(
Morgantown, WV). All other chemicals and solvents were
3
repeated twice. Then, the residue was dried under reduced
pressure for 1 min, and the reaction vessel was purged with a
flow of He (50 mL/min) for 1 min to ensure complete dryness.
Finally, the reaction vessel was cooled to room temperature and
used for further labeling.
analytical grade and were used without further purification
1
8
unless otherwise stated. [ F]Fluoride was produced with a
cyclotron (HM-18, Sumitomo Heavy Industries, Tokyo, Japan)
1
8
18
at Hamamatsu Photonics PET Center by the O(p,n) F nuclear
18
2
reaction using [ O]H O. Labeled compounds were synthesized
18
18
by using modified CUPID system (Sumitomo Heavy Industries,
Tokyo, Japan). The HPLC column used for the purification of
Preparation of Succinimidyl 4-[ F]fluorobenzoate ([ F]-
18
SFB). [ F]SFB was prepared according to the one-pot procedure
of Tang et al. (17) with some modifications. The precursor (5
18
18
succinimidyl 4-[ F] fluorobenzoate ([ F]SFB) was an Inertsil
ODS3 (10 × 250 mm, 5 µm, GL Sciences Inc., Tokyo, JAPAN).
Nonradioactive fluorine-conjugated siRNA was identified by
using a UPLC/ESI-QTOF-MS system, which consisted of an
ACQUITY ultraperformance liquid chromatography (UPLC)
system and an electrospray ionization quadrupole time-of-flight
mass spectrometer (SYNAPT High Definition Mass Spectrom-
etry system; Waters, Milford, MA, USA). An ultraviolet-visible
1
8
3
mg) in 1.5 mL of CH CN was added to the dried [ F]KF/
K[2,2,2] complex mentioned above and reacted at 80 °C for 10
min. After the reaction mixture had been cooled, 20 µL of 1 M
Pr
carried out at 120 °C for 5 min. Then, to the reaction mixture,
TSTU (15 mg) in 0.5 mL of CH CN was added and converted
to [ F]SFB at 80 °C for 5 min. The reaction mixture was diluted
with 2.0 mL of 5% CH COOH and transferred to the HPLC
injector. Crude product was purified by semipreparative HPLC
(CH CN:H O ) 300:700, 6 mL/min, 254 nm). The radioactive
peak eluted at 25.7 min was collected, diluted with 30 mL of
O, and passed through a Sep-Pak C18 cartridge (Waters).
4 3
NOH in 0.5 mL of CH CN was added; and hydrolysis was
3
18
(
UV/vis) detector (ACQUITY TUV, Waters) was used. The
3
UPLC column used for the identification of fluorine-labeled
siRNA was an Acquity UPLC BEH C18 column (2.1 × 100
mm, 1.7 µm, Waters Corp.). The particle size and zeta-potential
of nanoparticles were measured by using a Zetasizer Nano ZS
3
2
H
2
1
8
(
Malvern, Worcs, UK). Gel for obtaining an autoradiogram of
F and siRNA after electrophoresis was used an FLA-7000
[ F]SFB retained on the cartridge was released with 4 mL of
CH Cl and recovered into a V-vial by passage through a Sep-
Pak Dry cartridge (Waters). Then, the [ F]SFB was concen-
1
8
2
2
18
(
FUJIFILM Corporation, Tokyo, Japan) and LAS-3000 mini

758 Bioconjugate Chem., Vol. 21, No. 4, 2010
Hatanaka et al.
1
8
18
Scheme 1. Synthesis of the [ F]SFB and [ F]-Labeled siRNA
Table 1. Gradient Flow Table
Preparation of Cationic Liposomes and Their Complexes
with siRNA. DOTAP and cholesterol (1:1 as a molar ratio)
were dissolved in tert-butyl alcohol for freeze-drying and
hydrated in RNase-free PBS. The liposomes were frozen and
thawed for 3 cycles using liquid nitrogen and extruded 10 times
through a polycarbonate membrane filter having a pore size of
time (min)
0.1 M TEAA (%)
CH
3
CN (%)
flow rate (mL/min)
0-2
2-3
3-7
7-10
95-90
90-75
75-60
60-0
5-10
0.3
0.3
0.3
0.3
10-25
25-40
40-100
1
00 nm (Nucleopore, Maidstone, UK). Then, the cationic
trated by a flow of He (200 mL/min) at 60 °C and used for
liposome and siRNA were mixed gently and incubated for 10
min at room temperature to form liposome/siRNA complexes.
The nitrogen moiety of cationic liposome to the phosphorus of
siRNA (N/P ratio) was 24:1 in the complexes. The particle size
and zeta-potential of liposome/siRNA complexes diluted with
RNase-free PBS were measured. The particle size of liposome/
siRNA complexes was 218 ( 3.0 nm (n ) 3), and the particles
showed monodispersion (polydispersity index ) 0.16). The zeta-
potential was 36.3 ( 1.9 mV.
Near-Infrared Fluorescence Imaging in Vivo. The biodis-
tribution of AF750-siRNA was assessed by using an IVIS
Lumina System. Mice were fed an alfalfa-free feed to reduce
the effect of background fluorescence. The animals were
anesthetized continuously via inhalation of 2% escain. siRNA
or liposome/siRNA complexes containing 15 µg AF750-siRNA
were injected via a tail vein under anesthesia. Alexa Fluor 750
fluorescence was acquired every 10 min for up to 60 min after
the injection. After monitoring, the mice were sacrificed under
anesthesia. Then, the organs, namely, heart, lungs, liver, spleen,
and kidneys, were collected and imaged ex vivo with the IVIS.
18
labeling. [ F]SFB with (a specific radioactivity of 51.4 GBq/
µmol, a radiochemical yield of 21.1%) was obtained.
1
8
Radiolabeling of siRNA with [ F]SFB. siRNA (40 nmol)
in 16 µL of 50 mM borate buffer, pH 8.5, was added to the
1
8
[
F]SFB (60 nmol) in 3.6 µL of DMF. The mixture was
vortexed for a few seconds and then incubated at room
temperature for 20 min. The reaction mixture was purified and
concentrated by ultrafiltration through a 10 000 molecular weight
cutoff filter (Amicon, Millipore, Bedford, MA, USA). The
mixture was centrifuged at 4000 × g with RNase-free phosphate-
18
buffered saline (PBS). [ F]-labeled siRNA with a radiochemical
yield of 37.9% and a specific activity of 25.5 GBq/µmol was
obtained. Nonradioactive fluorine-conjugated siRNA was syn-
thesized in a similar manner except that 150 nmol cold SFB
1
8
was used instead of 60 nmol [ F]SFB.
Analytical Methods. Nonradioactive fluorine-conjugated
siRNA was identified by using a UPLC/ESI-QTOF-MS system.
The analytical column was maintained at 40 °C. A ultraviolet-
visible (UV/vis) detector, equipped with a 500 nL flow cell,
was also directly connected between the column outlet and the
QTOF-MS instrument. Fractionation of fluorine-conjugated
siRNA was performed at a flow rate of 0.3 mL/min using as
the eluent the gradient of 0.1 M triethylammonium acetate
18
PPIS Imaging of [ F]-Labeled siRNA. Biodistribution of
1
8
[ F]-labeled siRNA was imaged noninvasively by using a
PPIS (18-20). Animals were anesthetized with an intraperito-
neal injection of pentobarbital at 50 mg/kg, and then fixed on
an animal holder. To determine the whole-body biodistribution
(
TEAA, pH 7.0) and CH
the Table 1.
Evaluation of siRNA Labeling. Precursor siRNA, fluorine-
3
CN. Gradient profile is presented in
18
of siRNA, we administered the [ F]-labeled siRNA intrave-
nously (2.5 MBq/mouse). The scan was started immediately
after the administration and performed for 60 min. Images were
analyzed by using software ImageJ. After the PPIS scan, the
mice were sacrificed for the collection of the blood from a
carotid artery under anesthesia. Then, the heart, lungs, liver,
spleen, and kidneys were removed, and the biodistribution of
1
8
conjugated siRNA (non-RI), and [ F]-labeled siRNA (0.5 µg)
were applied to a 15% polyacrylamide gel and electrophoresed.
Then, the gel was exposed to an imaging plate for obtaining an
autoradiogram of F by using an FLA-7000; the gel was stained
for 5 min in ethidium bromide (EtBr), and siRNA was detected
by using a LAS-3000 mini system.
18

1
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Synthesis and PET Imaging of [ F]-Labeled siRNA
Bioconjugate Chem., Vol. 21, No. 4, 2010 759
18
Figure 2. Demonstration of [ F] labeling of siRNA. Precursor siRNA,
1
8
fluorine-conjugated siRNA, and [ F]-labeled siRNA were applied to
a 15% polyacrylamide gel, electrophoresed, and stained with ethidium
1
8
bromide. The gel was exposed to an imaging plate for detecting F.
18
basis of the result from non-RI fluorine-grafting to siRNA, [ F]-
labeled siRNA was prepared according to the same procedure,
and then electrophoresis assay and subsequent autoradiography
18
were performed to confirm the production of [ F]-labeled
siRNA. Electrophoresis assay with an imaging plate showed
18
that the siRNA was labeled with [ F]-fluorine without any other
exposure bands (Figure 2). In addition, the main band visualized
by EtBr staining showed the same position as the band on the
same polyacrylamide gel observed by autoradiography. As a
18
result, [ F]-labeled siRNA was successfully prepared without
any positron emitter-labeled byproducts, and the labeled siRNA
was not degraded under the experimental conditions used.
Biodistribution of siRNA Determined with near-Infrared
Fluorescence Imaging in Vivo. We examined the in vivo
behavior of siRNA by use of the NIRF imaging system. Mice
were intravenously administered naked AF750-siRNA or lipo-
some/AF750-siRNA complexes via a tail vein. The fluorescence
imaging of AF750-siRNA was started immediately after the
injection and monitored for 60 min. The biodistribution of
AF750-siRNA is shown in Figure 3. The results indicated that
the fluorescence compound was accumulated in the bladder
immediately after the administration of naked AF750-siRNA
and then excreted in the urine (Figure 3A). On the contrary,
fluorescence was observed at the upper part of the body where
the lung was positioned after the administration of liposome/
AF750-siRNA complexes. The total fluorescence intensity of
the image of mice injected with naked AF750-siRNA was
apparently higher than that obtained with the liposomal formula-
tion: Fluorescence in bladder after injection of naked siRNA
was quite intense because bladder is located near the body
surface, while that in lungs after injection of cationic liposome
complexes was weak because lungs are located more deep from
body surface compared with bladder.
Figure 1. UPLC analysis of fluorine-conjugated siRNA. Nonradioactive
fluorine was conjugated to siRNA and separated with the UPLC system.
(
A) Peak of precursor siRNA at 3.47 min. (B) Peaks of unreacted siRNA
at 3.47 min and fluorine-labeled siRNA at 3.87 min after the reaction.
[18
F]-labeled siRNA was measured with a gamma counter.
Distribution data were presented as percent dose per wet tissue.
The total volume of blood was assumed to be 7.56% of the
body weight. A time-activity curve was obtained from the mean
pixel radioactivity in the region of interest (ROI) of the PPIS
images.
Evaluation of siRNA Stability against Serum-Mediated
Degradation. Fluorine-conjugated (0.5 µg) naked siRNA or that
complexed with liposomes was incubated in 90% fetal bovine
serum for 60 min at 37 °C. The siRNA was extracted from the
serum by using Trizol reagent and subjected to 15% polyacryl-
amide gel electrophoresis. The gel was stained for 5 min in
EtBr, and siRNA was detected by using a LAS-3000 mini
system.
The ex vivo imaging showed that a small amount of
fluorescence remained in the kidneys but that no fluorescence
was detected in the other tissues examined (Figure 3B). In
contrast, the fluorescence of the cationic liposome/AF750-siRNA
complexes accumulated in the lungs, being consistent with the
in vivo imaging data.
RESULTS
Stability of Fluorine-Conjugated siRNA against Serum-
Mediated Degradation. We evaluated the stability of naked
fluorine-conjugated siRNA and cationic liposome/fluorine-
conjugated siRNA complexes in serum for 60 min (Figure 4).
The naked fluorine-conjugated siRNA incubated in PBS (con-
trol) was not decomposed. However, the band of naked siRNA
obtained by electrophoresis of the sample that had been
incubated in the presence of serum disappeared, indicating that
the siRNA had been degraded by RNase within 60 min. In
contrast, the band of siRNA was detected in the case of the
cationic liposome/siRNA complexes exposed to the serum,
indicating that complex formation protected siRNA from RNase
in the serum.
[18
F]-Labeling of siRNA and Its Radiochemistry. The
1
8
18
methods for synthesis of [ F]SFB and [ F]-labeling of siRNA
are shown in Scheme 1. At first, we prepared non-radioisotope
non-RI) fluorine-conjugated siRNA and identified the com-
pound by using LC/ESI-TOF-MS (Table 1). Unreacted siRNA
showed a peak at 3.47 min (Figure 1A). After the reaction,
HPLC analysis indicated 2 main peaks, namely, labeled siRNA
at 3.87 min and precursor siRNA at 3.47 min, respectively
(
(
Figure 1B). These peaks were fractionated by HPLC and
analyzed by ESI-TOF-MS, giving multiply charged ion peaks
of m/z [M-8H]8 2196.1, [M-7H] 2510.9, and [M-6H]
-
7-
6-
2
930.0 (data not shown), which corresponded to the labeled
8
-
7-
siRNA (expected mass of m/z [M-8H] 2196.7, [M-7H]
In Vivo PPIS Imaging of siRNA Trafficking. Next, we
-
18
2
510.7, and [M-6H]6 2929.3). The overall yield of labeled
examined [ F]-labeled siRNA trafficking by use of PPIS. Mice
18
siRNA was greater than 30% based on the peak area. On the
were intravenously administered naked [ F]-labeled siRNA in

760 Bioconjugate Chem., Vol. 21, No. 4, 2010
Hatanaka et al.
Figure 3. NIRF in vivo imaging with AF750-siRNA and liposome/AF750-siRNA complexes. (A) AF750- siRNA (top) or liposome/AF750-siRNA
complexes (bottom) were intravenously administered to BALB/c mice. Images were acquired every 10 min for up to 60 min after the administration.
The white arrowhead shows the bladder region, and the yellow arrowhead, the lung region. (B) Ex vivo imaging of AF750-siRNA and liposome/
AF750-siRNA complexes at 60 min after administration.
1
8
18
F after administration of naked [ F]-labeled siRNA had not
been distributed in any tissues tested, whereas the radioactivity
18
after the injection of the cationic liposome/[ F]-labeled siRNA
complexes was highly distributed in the lungs (Figure 5C). These
results corresponded roughly to the data obtained from the
fluorescence ex vivo imaging.
Moreover, to confirm whether the accumulation of 18F in the
18
lungs after administration of cationic liposome/[ F]-labeled
siRNA reflected the accumulation of the liposomes of the
complex, we examined the biodistribution of cationic liposome/
Figure 4. Stability of naked siRNA or liposome/siRNA complexes in
FBS. siRNA (0.5 µg) was incubated with PBS (-) or 90% FBS at 37
°
C for 60 min. siRNA was extracted and electrophoresed on a 15%
polyacrylamide gel after a 60 min incubation and stained with EtBr.
Untreated siRNA was used as a control marker.
3
siRNA complexes prepared with [ H]cholesterylhexadecyl ether
as a component. As a result, the liposomal complexes with
siRNA were distributed in the lungs quite similarly to the
distribution of the F, suggesting that cationic liposome/siRNA
accumulated in lungs as the complex form (Supporting Informa-
tion Figure S1).
1
8
18
PBS (-) or liposome/[ F]-labeled siRNA complexes via a tail
1
8
vein. The real-time imaging of [ F]-labeled siRNA using PPIS
was acquired for 60 min, and images were integrated every 5
1
8
18
min (Figure 5A). The F radioactivity of the naked [ F]-labeled
siRNA accumulated inside the kidneys immediately after
administration. This accumulation was not detected in NIRF in
vivo imaging because of the influence of tissue depth. Then,
DISCUSSION
Recently, the topical application of siRNA medicines has
reached clinical trial, and siRNA delivery systems for systemic
injection have been extensively studied for the next generation
of siRNA medicines (21). Pharmacokinetic information on
siRNA molecules is considered to be particularly important for
the acceleration of the development of siRNA medicine. Among
the technologies for pharmacokinetic analysis, PET imaging
technique is considered to be applicable to both preclinical trial
and microdosing study (human phase 0 study). Using subtoxic
and subpharmacologic doses, PET microdosing studies can be
performed to screen for drug candidates for clinical trials on
the basis of their pharmacokinetic properties (22, 23).
1
8
F was transferred to the bladder and subsequently excreted in
the urine. In contrast, most of the 1 F radioactivity after the
8
1
8
administration of cationic liposome/[ F]-labeled siRNA com-
plexes were retained in the lungs for 60 min, and only
insignificant accumulation of them was observed in the bladder.
The time-activity curve of F in naked siRNA showed transient
accumulation in kidney immediately after injection (19%
injected dose/tissue at 2 min), and half of that was transferred
to bladder at 21 min, and then the excretion of F to bladder
was gradually increased (Figure 5B). In contrast, the most of
the F after injection of liposome/siRNA complexes im-
mediately accumulated in lungs and was maintained up to 60
min (76% injected dose/tissue). The biodistribution data obtained
with a gamma counter after organ dissection showed that the
18
1
8
1
8
In the present study, we labeled double-stranded siRNA
18
with F to obtain pharmacokinetics information about siRNA
18
and siRNA in DDS carriers. We used [ F]-fluorine as a short-

1
8
Synthesis and PET Imaging of [ F]-Labeled siRNA
Bioconjugate Chem., Vol. 21, No. 4, 2010 761
18
18
Figure 5. PPIS imaging with [ F]-labeled siRNA and liposome/[ F]-labeled siRNA complexes. (A) Naked siRNA (top) or liposome/siRNA complexes
bottom) at 2.5 MBq in 0.2 mL were intravenously administered to BALB/c mice. Images were acquired with a 1 min frame at 1, 10, 20, 30, 40,
0, and 60 min after the administration. The green arrowhead shows kidneys; the white one, the bladder; the red one, urine; and the yellow one,
(
5
1
8
18
18
the lungs. (B) Real-time changes in [ F]-labeled siRNA and liposomal [ F]-labeled siRNA. Time activity curves of F with naked siRNA shows
1
8
kidney (blue) and bladder (red) and that of F with cationic liposome/siRNA shows lung (black) and bladder (green). (C) After the PPIS scan, the
mice were sacrificed, and biodistribution of F in each organ was then measured with a gamma counter.
1
8
lived positron-emitting radionuclide (half-life: 109 min), which
has been applied to humans for cancer diagnosis in the form of
We speculate that siRNA was degraded in the liver and excreted,
while cholesterylhexadecyl ether did not.
1
8
18
[
2- F]2-deoxy-2-fluoroglucose ([ F]FDG). The advantages of
In the present study, we first used AF750-siRNA and
examined its in vivo behavior by using NIRF fluorescence
imaging. NIRF fluorescence was rapidly cleared from the
bloodstream and excreted in the bladder after the administration
of naked AF750-siRNA (Figure 3). In contrast, the fluorescence
of cationic liposome/siRNA complexes accumulated in the
lungs. However, the differential fluorescence intensities between
in vivo images and ex vivo ones indicated the limitation of NIRF
imaging. Quantitative analysis could not be applied due to the
influence of tissue depth on the fluorescence intensity. Moreover,
it is possible that fluorescence self-quenching and/or resonance
energy transfer affect the intensity.
the present methodology to label siRNA are that (1) the
modification of double-stranded RNA with amino group enables
fast preparation and purification without the time-consuming
process of annealing and (2) the methodology maintains the
conformational accuracy of the siRNA. Indeed, reaction of
18
siRNA and [ F]SFB was complete within 20 min. In addition,
fluorine-conjugated siRNA was identified by LC and ESI-TOF-
MS (Figure 1) and did not decompose during the reaction or
purification process (Figure 2).
It is well-known that one of the most important factors
regarding the biodistribution of liposomes in vivo is the charge
of the liposomal surface. Positively charged complexes ag-
gregate in the presence of serum proteins (24), are entrapped
in the lung capillaries, and thus accumulate in the lung
tissue (25, 26) after intravenous administration. In fact, when
In contrast to NIRF imaging, PPIS or PET technology
provides quantitative analytic data with high spatial resolution.
1
8
F was observed in the kidneys until 10 min after the
18
3
administration of naked [ F]-labeled siRNA, and subsequently,
it was transferred to the bladder and excreted into urine (Figure
liposomes were labeled with [ H]cholesterylhexadecyl ether, the
radioactivity of cationic liposome/siRNA complexes accumu-
lated in the lungs (Supporting Information Figure S1). However,
since nucleic acids such as siRNAs and oligodeoxynucleotides
5
). This result is consistent with previous result reported by
18
Viel et al. who used F-labeled naked siRNA (11). Other reports
also presented the renal excretion of siRNA (27, 29, 30). In
(
ODNs) are known to be eliminated from the circulation via
64
addition, Bartlett et al. reported that the excretion of Cu-labeled
naked siRNA was quite fast (9). They indicated that the siRNA
in a complex with cyclodextrin-containing polycation excreted
quite rapidly and suggested the instability of the complex.
Therefore, determination of siRNA trafficking instead of carrier
trafficking is important for the development of siRNA medi-
the kidneys and excreted into the urine 60 min after administra-
tion via a tail vein (27, 28), distribution studies on siRNAs or
ODNs besides on their carriers are important for the develop-
ment of nucleic acid medicines. In the distribution study,
3
[
H]cholesterylhexadecyl ether was accumulated in the liver to
some extent, although the accumulation of siRNA was not
observed after injection of cationic liposome/siRNA complex.
1
8
cines. In contrast to the in vivo fate of naked siRNA, the F of

762 Bioconjugate Chem., Vol. 21, No. 4, 2010
Hatanaka et al.
1
8
cationic liposome/[ F]-labeled siRNA complexes was spread
throughout the lungs and was retained in them at least up to 60
min.
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PEG-LHRH conjugate and PEI. Bioconjugate Chem. 19, 2156–
Next, we investigated the stability of siRNA in the presence
of serum by performing an electrophoresis assay. The data
indicated that naked siRNA was degraded when incubated for
(
6
0 min in serum, whereas the liposome/siRNA complexes were
not (Figure 4). These results suggest that naked siRNA has a
short half-life in vivo and is rapidly eliminated by renal excretion
as a degraded form. However, further study is needed to clarify
2
162.
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medicines.
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ACKNOWLEDGMENT
This study was financially supported by the Health and Labor
Sciences Research Grants from the Ministry of Health, Labour,
and Welfare of Japan. Synthesis of positron emitter-labeled
siRNA was supported in part by Hokkaido System Science Co.
Ltd. We thank Drs. T. Kakiuchi and H. Uchida at Hamamatsu
Photonics K.K. PET Center for PET study for their technical
assistance. They also thank Drs. S. Akai and T. Toyo’oka at
the University Shizuoka for their valuable discussions as well
as for allowing us to use their facilities. We also acknowledge
Dr. S. Inagaki for helpful discussions and excellent technical
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