Hybrid PET/CT for noninvasive pharmacokinetic evaluation of dynamic polyconjugates, a synthetic siRNA delivery system

Article abstract of DOI:10.1021/bc900558z

Positron emission tomography/computed tomography (PET/CT) hybrid imaging can be used to gain insights into a synthetic siRNA delivery system targeted to the liver. Either siRNA or the delivery vehicle was labeled with ⁶⁴Cu via 1, 4, 7, 10- tetraazacyclododecane- 1, 4, 7, 10- tetraacetic acid (DOTA) chelation. This study confirmed that the siRNA delivery system was successfully targeted to the liver. Incorporation of the siRNA into the delivery system protected the siRNA from renal filtration long enough so that the siRNA could be delivered to the liver. PET/CT imaging was important for confirming biodistribution and for determining differences in the distribution of labeled siRNA, siRNA incorporated into the delivery system, and the delivery system without siRNA.

Full text of DOI:10.1021/bc900558z

Bioconjugate Chem. 2010, 21, 1183–1189
1183
Hybrid PET/CT for Noninvasive Pharmacokinetic Evaluation of Dynamic
PolyConjugates, a Synthetic siRNA Delivery System
Sarah R. Mudd,† Vladimir S. Trubetskoy,*,‡ Andrei V. Blokhin,‡ Jamey P. Weichert,§ and Jon A. Wolff‡
Department of Pharmaceutical Sciences, University of Wisconsin - Madison, 777 Highland Ave., Madison, Wisconsin 53705,
Roche Madison Inc., 465 Science Dr., Madison, Wisconsin 53711, Department of Radiology, University of Wisconsin -
Madison, 600 Highland Ave., Madison, Wisconsin 53792. Received December 15, 2009; Revised Manuscript Received May 25, 2010
Positron emission tomography/computed tomography (PET/CT) hybrid imaging can be used to gain insights into
a synthetic siRNA delivery system targeted to the liver. Either siRNA or the delivery vehicle was labeled with
64Cu via 1, 4, 7, 10- tetraazacyclododecane- 1, 4, 7, 10- tetraacetic acid (DOTA) chelation. This study confirmed
that the siRNA delivery system was successfully targeted to the liver. Incorporation of the siRNA into the delivery
system protected the siRNA from renal filtration long enough so that the siRNA could be delivered to the liver.
PET/CT imaging was important for confirming biodistribution and for determining differences in the distribution
of labeled siRNA, siRNA incorporated into the delivery system, and the delivery system without siRNA.
using a similar procedure. While hybridization- and PCR-based
methods have been used to track siRNA (5), such an approach
would not be applicable for following the distribution of a
synthetic polymer such as PBAVE. Also, a hybridization or
PCR-based method only detects relatively intact siRNA, while
a label-based method can assess total accumulation indepen-
dently of the intactness of the siRNA over a certain time period,
assuming slow removal of the label from the target tissue.
Furthermore, a quantitative imaging procedure provides ad-
ditional information about distribution that would not be
available from procedures that rely upon the use of tissue
extracts. A disadvantage of a label-based procedure is that parent
and metabolites cannot be distinguished in ViVo. To address this
concern somewhat, a chemically modified siRNA sequence that
was relatively resistant to nuclease degradation was used. In
addition, a tissue imaging procedure cannot delineate the
subtissue cellular distribution and must be coupled with
microscopy studies. Our previous study using confocal micros-
copy showed that the DPCs delivered fluorescently labeled
siRNA predominantly to hepatocytes within the liver (3).
Prior siRNA in ViVo imaging studies have used PET, SPECT,
and planar gamma camera imaging. Labeling of siRNA has been
achieved using the positron emitters 18F and 64Cu; SPECT has
been done using 125I, 111In, and 99m Tc (6-11). The first report
of siRNA delivery assessment using nuclear imaging was done
using PET with 64Cu chelated by DOTA (6). Planar gamma
camera imaging techniques were ineffective for quantitative
biodistribution studies because internal organs overlap in the
same plane making the precise uptake in each unknown. For
example, liver and kidney activities were reported together as
a sum (8). Because siRNA kinetics are very rapid, an imaging
technique must be chosen that is quantitative and has good
temporal resolution. PET has superior sensitivity compared to
SPECT, which allows for improved image quality as well as
improved temporal resolution. This allows for many short frames
to be obtained in a dynamic image series (12). Thus, we chose
PET imaging for our studies.
INTRODUCTION
The delivery of short interfering RNA (siRNA) into cells is
a powerful approach to selectively inhibit gene expression (1).
For therapeutic application in humans, a variety of approaches
are being developed to deliver the siRNA to the appropriate
target cells in ViVo. These include siRNA-conjugates and a
variety of lipid- and polymer-based delivery systems (2). We
recently developed a new polymer-based delivery system,
termed dynamic polyconjugates (DPCs), for delivering siRNA
to hepatocytes and other cells in ViVo (3). A key element of
this new approach is the use of masked endosomolytic poly-
mers that improve the release of nucleic acids from endosomes.
When the masked endosomolytic polymers enter the acidic
environment of the endosome, a pH-labile bond is broken,
releasing the agent’s endosomolytic capability. This approach
enables polyethylene glycol (PEG) and targeting ligands to be
attached to the polymer without disturbing its endosomolytic
function, while still enabling in ViVo delivery and targeting. In
its current version for hepatocyte delivery, the endosomolytic
polymer is an amphipathic poly vinyl ether composed of butyl
and amino vinyl ethers termed PBAVE. The asialoglycoprotein
receptor ligand N-acetylgalactosamine (NAG) is used to target
the DPC to hepatocytes. PEG is used to discourage nonspecific
interactions. Both the NAG and the PEG are attached to the
PBAVE via pH-labile bonds. The siRNA is covalently attached
to the PBAVE via a labile disulfide bond. The resulting DPC
is 10 ( 2 nm (3).
We were interested in studying the tissue distribution and
kinetics of DPCs in mice. Advances in animal and human
imaging techniques have made it possible to refine the process
of modern drug discovery, specifically in assessing the delivery
of an experimental drug to its targeted and nontargeted tissues
(4). Of particular interest was evaluating the distribution of both
the endosomolytic polymer PBAVE and siRNA components
* Corresponding Author: Vladimir S. Trubetskoy, Address: 465
Science Dr., Madison, WI 53711. E-mail: Vladimir.trubetskoy@roche.com,
Telephone number: (608)316-3925, Fax number: (608)441-0741.
† Department of Pharmaceutical Sciences, University of Wisconsin -
Madison.
We designed the present study to examine the whole body
biodistribution of therapeutically relevant doses of DPCs
containing siRNA targeted to hepatocytes in ViVo using micro-
PET/CT hybrid imaging with 64Cu DOTA-labeled DPCs. The
half-life of 64Cu is 12.7 h, which is reasonable for imaging
‡ Roche Madison Inc.
§ Department of Radiology, University of Wisconsin - Madison.
10.1021/bc900558z  2010 American Chemical Society
Published on Web 06/16/2010

1184 Bioconjugate Chem., Vol. 21, No. 7, 2010
Mudd et al.
alkylation of amine 3 with 4-[(N-(2-chloroethyl)-N-methyl]ami-
nobenzaldehyde 2 followed by Boc-deprotection with CF3CO2H
(Figure 1) (14, 15). Aldehyde 2 was prepared from 4-[N-(2-
hydroxyethyl)-N-methyl]aminobenzaldehyde 1 (27.9 mmol) by
reaction with triphenylphospine (58.6 mmol) in CCl4/CHCl3 (2:
1, 75 mL) mixture under Ar at 55 °C for 1 h. The solvent was
evaporated in Vacuo and the product 2 was purified on a silica
gel column (hexane:EtOAc 8:2). The structure was verified by
1H NMR.
N-(3-aminopropyl)-N-[3-[[[4-[(2-chloroethyl)methylamino]phe-
nyl]methyl]amino]propyl]-N,N-dimethylammonium tetrakis(tri-
fluoroacetate) 4 (0.096 mmol) was dissolved in DMF before
being combined with DOTA-NHS-ester 5 (0.1 mmol) and
diisopropylamine (0.46 mmol). The mixture was stirred for 4 h
at 20 °C under Ar. The product, N-(3-DOTA-amidopropyl)-N-
[3-[[[4-[(2-chloroethyl)methylamino]phenyl]methyl]amino]propyl]-
N,N-dimethylammonium trifluoroacetate 6 was precipitated in
10 mL of Et2O and purified by HPLC on a Gemini C-18, 5
µm, 110 Å column, 260 × 21.2 mm (Phenomenex). The mobile
phase was CH3OH(TFA 0.1%)-H2O(TFA 0.1%), 15 mL/min
and the organic gradient was 12% (4.5 min), 12-32% (25 min),
32-95% (5 min), 95% (10 min), 90-12% (1 min). UV
monitoring was done at 210 nm.
Modification of siRNA. The siRNA used with both the DOTA-
PBAVE DPC and DOTA-siRNA experiments was modified
with succinimidyl-S-acetylthioacetate (SATA) as previously
described by Rozema, et al. (3). To make DOTA-siRNA, the
siRNA was treated with DOTA-NM (2:1 w/w) in 5 mM TAPS,
pH 9.0 for 2 h at 37 °C. The final product was purified from
the excess of chelate by 3x ethanol precipitation.
Preparation of DOTA-PBAVE. PBAVE polymer was modi-
fied with DOTA-NHS ester by incubating the polymer and the
activated ester in 20 mM HEPES buffer, pH 8.0 for 1 h at room
temperature. Polymer concentration was 10 mg/mL, molar ratio
of the activated ester to the polymer amines was 2%. The
modified polymer was purified using QAE-Sephadex column
in water and freeze-dried.
64Cu labeling. The 64Cu chloride was mixed with 0.5 M
sodium acetate buffer at pH 5.5 and DOTA-siRNA or activated
DOTA-PBAVE at a ratio of approximately 4 mCi:75 µg DOTA-
siRNA or 500 µg activated DOTA-PBAVE. The siRNA mixture
was incubated at 40 °C for one hour; if activated DOTA-PBAVE
was used, the mixture was incubated at room temperature for
30 min. To remove the unconjugated 64Cu, size exclusion
chormatography was performed using Sephadex G-50 columns
for siRNA labeled product and QAE columns for PBAVE
labeled product. Centrifugation was performed in a Beckman
TJ-6 centrifuge (Fullerton, CA) for 2 min at 2000 rpm.
Dynamic Polyconjugate Synthesis and Formulation. The
polyconjugate was synthesized and formulated according to
previously published procedures at an siRNA/PBAVE ratio of
1:10 (w/w) (3). A ratio of 2:1 of PEG:NAG was used to modify
the PBAVE, which contains no more than one siRNA covalently
attached. To remove unconjugated siRNA, a Sephadex G-75
column was used for 5 min at 1000 rpm.
Radio-Thin Layer Chromatography Studies. Radio-TLC stud-
ies were performed using EMD Merck Silica Gel 60 TLC plates
and methanol: 5% ammonium acetate (1:1 v/v) mobile phase.
The plates were scanned using Bioscan AR 2000 Imaging
Scanner (Washington, DC).
Animal studies. Female, ICR mice were obtained from Harlan
(Indianapolis, IN). The number of animals per group and naming
scheme used in the figures is as follows: n ) 3 for free 64Cu
(64Cu), n ) 4 for 64Cu DOTA siRNA (labeled siRNA), n ) 4
for 64Cu DOTA siRNA DPC (DPC), and n ) 3 for 64Cu DOTA
PBAVE/DPC (PBAVE/DPC). All animal studies were per-
formed in accordance with all guidelines and were approved
Figure 1. Preparation of DOTA-NM.
siRNA kinetics. Because the DPC system consists of two main
components, the siRNA (payload) and an excess of the polymer
PBAVE (carrier), the primary objective was to image the
kinetics of tissue distribution of both components in ViVo. This
study also incorporated several modifications of existing
procedures. A new DOTA-labeling reagent was synthesized that
enables the attachment of the chelated 64Cu to siRNA after its
synthesis. A CT contrast reagent was utilized to ensure accurate
liver definition and coregistration with the PET scans. In
addition, we used a stabilized siRNA containing 2′O-methyl
modified nucleotides and phosphorothioate linkages in this study
(13), while previous imaging studies used less stabilized
siRNAs (6, 8, 10).
MATERIALS AND METHODS
Materials. Primary amine modified ApoB-2 siRNA sequence
(13) was ordered from Ambion (Austin, TX) (13). DOTA-NHS-
ester 5 was purchased from Macrocyclics (Dallas, TX). 4-[N-
(2-Hydroxyethyl)-N-methyl]aminobenzaldehyde 1 (Figure 1)
was purchased from Sigma-Aldrich. Compounds 3 and 4 (Figure
1) were prepared by a known procedure (14). 64Cu chloride was
provided by the University of Wisconsin cyclotron facility
(Madison, WI). Sephadex G50, G75 and QAE columns were
purchased from GE Healthcare (Piscataway, NJ).
Methods. Preparation of DOTA-nitrogen mustard (DOTA-
NM). In preparation of precursor 4 we utilized reductive

Hybrid PET/CT for Evaluation of Dynamic PolyConjugates
Bioconjugate Chem., Vol. 21, No. 7, 2010 1185
time of injection and acquired for one hour post-injection and
reconstructed using an OSEM 2D reconstruction algorithm.
Listmode data were binned into sinograms of dimensions 1, 4,
10, 25, 45, and 75 s frames and 2, 4, 8, 12, 20, 30, 40, 50, and
60 min frames; all frames were decay corrected during
reconstruction. CT scans were acquired at 80 kVp and 1000
µA and reconstructed using a filtered back projection algorithm
with a Shepp-Logan filter. Scatter correction and attenuation
correction, based on CT imaging, were done for all PET images.
Image analysis and registration was performed using Inveon
Research Workplace (IRW) (Knoxville, TN). All images
displayed have the same PET window leveling to ensure
accurate comparison. All regions of interest (ROIs) were drawn
in the CT field of view to ensure accurate organ definition; CT
contrast was utilized to ensure accurate liver definition. The
injected dose was determined by creating a 3D ROI for the entire
mouse. Ellipsoid regions of interest were drawn over the liver,
kidneys, bladder, heart, spleen, and lungs to obtain tracer 64Cu
concentration values in units of µCi/mL, which were normalized
to percent injected dose per mL. One region of interest per organ
was drawn to include the maximum volume possible without
including nonROI organ tissue. Percent injected dose per organ
(%ID) values were calculated based on average organ volumes
(16). The organ volumes for a 20 g animal were scaled based
on the weight of the animals used. Average values with standard
deviation are reported.
Figure 2. Schematic representations of (A) Labeled 64Cu (64Cu), (B)
64Cu DOTA siRNA (Labeled siRNA), (C) 64Cu DOTA siRNA DPC
(DPC), and (D) 64Cu DOTA PBAVE:DPC (PBAVE/DPC).
by the University of Wisconsin-Madison and Roche Madison
animal care and use committees.
In ViVo imaging using microPET/CT. All images were
acquired on a Siemens Inveon microPET/CT scanner (Knoxville,
TN). Approximately 3 h prior to the start of the CT scan, mice
were injected with 120 µL of eXIA 160 (Binitio, Ottawa,
Canada) liver CT contrast agent in order to accurately define
the liver. Mice were anesthetized using 1 mg/kg body weight
of medetomidine and 75 mg/kg body weight of ketamine prior
to the injection of radiotracer and imaging procedures. Between
250 - 300 µCi, 100 - 250 µL, of the selected 64Cu labeled
agent was injected via catheter in the tail vein while the animal
was on the scanner. Dynamic PET scans were started at the
3D MoVies. 3D movies were made using Amira (San Diego,
CA) software for one representative animal per group. The 3D
movies show the CT image with the dynamic PET data (see
online Supporting Information).
RESULTS AND DISCUSSION
64Cu Labeling. The 64Cu chelator DOTA-NHS ester has
shown to be a convenient method of labeling synthetic polymers
for testing various bioconjugates in ViVo (17). The DOTA moiety
(1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid)
Figure 3. PET/CT images of (A) free 64Cu and (B) 64Cu-labeled siRNA (Labeled siRNA) at 60 min post-injection and corresponding time-activity
curves for the (C) liver and (D) kidneys shown as percent injected dose per mL and (E) liver and (F) kidney shown as percent injected dose.

1186 Bioconjugate Chem., Vol. 21, No. 7, 2010
Mudd et al.
chelates 64Cu while the N-hydroxysuccinamide (NHS) moiety
reacts with amines. Accordingly, this strategy was chosen for
labeling the primary amines of PBAVE with 64Cu. In order to
attach the DOTA group to siRNA, the nucleic acid-reactive
DOTA-based chelation reagent, DOTA-NM, was synthesized
(Compound 6, Figure 1). It contains the DOTA chelator and
an alkylating moiety that reacts with nucleic acid bases
randomly. Both labeling procedures were carried out in 2%
DOTA, which corresponds to 0.75 mol DOTA/mol siRNA.
The incorporation of 64Cu into DOTA was performed using
a standard method of transchelation from slightly acidic acetate
or citrate buffers at elevated temperatures. The purification of
the labeled species prior to injection was performed by
centrifugation on G50 or QAE columns. Successful labeling of
both siRNA and PBAVE was confirmed by labeling with 64Cu
in which approximately 50 - 85% of 64Cu was conjugated to
DOTA siRNA or DOTA PBAVE. It was confirmed that free
64Cu remained in the column after centrifugation by measuring
radioactivity in a dose calibrator.
The stability of the 64Cu DOTA complex on PBAVE modified
with PEG and NAG was confirmed in Vitro using radioTLC
analysis. It is possible that the carboxyl groups on the carboxy
dimethylmaleic anhydride (CDM) derivatives, containing PEG
or NAG, could potentially chelate the free 64Cu or strip the 64Cu
from the DOTA. However, there was no association of free 64Cu
with CDM-modified PBAVE without the presence of DOTA
on the PBAVE. 64Cu remained bound to the DOTA-modified
PBAVE (data not shown).
The tissue distribution of a variety of 64Cu-labeled molecules
and DPC complexes was studied and is depicted in Figure 2.
These include free 64Cu (64Cu; Figure 2A), siRNA (labeled
siRNA; Figure 2B), 64Cu-labeled siRNA within DPC complexes
containing mostly siRNA covalently linked to the PBAVE
polymer (DPC; Figure 2C), and 64Cu-labeled PBAVE within
unpurified DPC complexes (PBAVE/DPC Figure 2D). The
percent conjugation of siRNA to PBAVE was not measured
for these experiments, but values typically range from 70 -
90% of the input siRNA (3).
Free 64Cu Imaging Studies. Given the possible dissociation
of free 64Cu from the polymer and siRNA complexes, the tissue
distribution of free 64Cu, unassociated with any other molecule,
was determined (Figure 3). Free 64Cu was mainly taken up by
the liver after intravenous administration (Figure 3A) in
agreement with a previous report (6). The concentration in the
liver steadily increased and then reached a plateau approximately
30 min after administration (Figure 3C and E). Liver-associated
radioactivity for all experiments was confirmed by coregistration
of the PET and CT images; the liver was contrast enhanced in
the CT. At one hour post-injection 25.1 ( 5.3% injected dose/
mL or 38.6 ( 7.8% injected dose of free 64Cu was present in
the liver. The only organ, other than the liver, with substantial
amount of radioactivity was the kidney (Figure 3D and F). The
peak concentration of 64Cu in the kidneys was 25.9 ( 14.2%
injected dose/mL at 1.25 min postinjection. At one hour post-
injection only 4.3 ( 2.6% of the total injected dose was present
in the bladder. Because very little of the 64Cu was present in
the bladder and the peak in kidney concentration was seen soon
after injection, the peak concentration seen in the kidneys at
1.25 min post-injection was most likely due to 64Cu in the blood
and not in the kidney tissue. Free copper normally enters
hepatocytes to be used in the synthesis of proteins in the liver
(ceruloplasmin) or to be eliminated via biliary excretion (18).
Figure 4. Time activity curves for (A) kidney as percent injected dose
per milliliter and (B) kidney as percent injected dose, and (C) urine as
percent injected dose for 64Cu-labeled siRNA (labeled siRNA), 64Cu
DOTA siRNA DPC (DPC), and 64Cu DOTA PBAVE/DPC (PBAVE/
DPC).
free 64Cu. However, the peak amount of labeled siRNA in the
liver was 32.9 ( 19.8% of the total injected dose, which
occurred 45 s after injection. It cannot be determined whether
this is due to siRNA in the blood or actual uptake and
degradation in the liver tissue. However, considering the timing
of the peak, a large fraction of this was most likely due to labeled
siRNA that was still in the blood.
64Cu-Labeled siRNA (Labeled siRNA) Imaging Studies.
The distribution of 64Cu-labeled siRNA was very different from
that of free 64Cu (Figure 3). At the end of 60 min, labeled siRNA
accumulated much less in the liver: less than 10% of the injected
dose was present in the liver as compared to almost 40% with
In comparison to free 64Cu, more of the labeled siRNA-
associated radioactivity appeared in the kidneys, especially at
the early time points (Figure 3B,D,F). The maximum labeled

Hybrid PET/CT for Evaluation of Dynamic PolyConjugates
Bioconjugate Chem., Vol. 21, No. 7, 2010 1187
Figure 5. PET/CT images of (A) naked 64Cu DOTA siRNA (siRNA), (B) 64Cu DOTA siRNA DPC (DPC), and (C) 64Cu DOTA PBAVE/DPC
(PBAVE/DPC) at 60 min post-injection and corresponding time-activity curve for the liver as (D) percent injected dose per milliliter and (E)
percent injected dose.
siRNA seen in the kidneys was 56.9 ( 10.8% injected dose/
mL or 28.3 ( 2.5% of the injected dose at 4 min post-injection.
At later times, the amount of radioactivity decreased in the
kidneys and accumulated in the bladder (Figures 4A,B,C and
5A). At one hour after injection, 62.9 ( 8.7% of the injected
dose was present in the bladder (Figure 4C).
(Figures 4 and 5B,D,E). A rapid peak followed by a plateau
was observed in the liver. One hour after injection of DPC,
38.4 ( 6.0% injected dose/mL or 70.1 ( 2.5% of the injected
dose accumulated in the liver.
With the siRNA labeled DPC preparation, the concentration
in the kidney peaked 8 min after injection at 19.1 ( 3.1%
injected dose/mL or 9.3 ( 2.0% injected dose. At one hour
post-injection, 10.1 ( 6.1% of the radioactivity was present in
the bladder.
This is the highest siRNA delivery efficiency to the liver yet
reported. Kissel et al. reported 15% of the injected dose of PEI/
siRNA complexes delivered to the liver, and Morrissey et al.
noted 28% of the injected dose of siRNA delivered to the liver
using stable nucleic acid-lipid particles (SNALP) (8, 19). With
both of these studies, the delivery vehicle is not targeted to a
specific cell type. For delivery to the liver, SNALP relies on
the liver’s ability to clear particles of a certain size, and uptake
by Kupffer cells is observed (20). Thus, it is likely that a
significant portion of the siRNA delivered using PEI and
SNALP is not taken up by hepatocytes. In contrast, we have
previously demonstrated using confocal microscopy that the
majority of the liver uptake following DPC siRNA delivery is
Using imaging techniques to study biodistribution, Davis et
al. and Braasch et al. reported uptake of siRNA by the liver
and kidney in the mouse one hour postinjection with more
activity being present in the liver (6, 11). However, van de Water
et al. reported minimal uptake of siRNA by the liver compared
to the kidneys in rats. The differences in van de Water et al.’s
reported values and Braasch et al.’s were attributed to the
sequence of siRNA, labeling method, or differences in species
(10). Since the labeling method presented in this paper is similar
to the method used by Davis et al., the most likely explanation
for the differences in liver uptake may be due to differences in
chemical modification, the sequence of siRNA, or differences
in animal breeds.
64Cu-Labeled siRNA DPC (DPC) Imaging Studies. With
the DPC preparation, a much larger percentage of the injected
siRNA accumulated in the liver and a smaller percentage
accumulated in the bladder compared with labeled siRNA

1188 Bioconjugate Chem., Vol. 21, No. 7, 2010
Mudd et al.
in hepatocytes (3). This is consistent with receptor ligand
mediated uptake of DPC.
free siRNA, DPC with the 64Cu-labeled PBAVE and DPC with
the 64Cu-labeled conjugated siRNA were prepared as indicated
in Materials and Methods section. This material is available free
of charge via the Internet at http://pubs.acs.org.
64Cu-Labeled PBAVE/DPC (PBAVE/DPC) Imaging Stud-
ies. The DPC contains an excess of the NAG-targeted PBAVE
not conjugated with siRNA. Thus, the tissue distribution 64Cu-
labeled PBAVE (Figures 4 and 5) is of interest in order to gain
insight into how the PBAVE behaves in ViVo. One hour after
injection, 44.7 ( 2.7% injected dose/mL or 97.9 ( 12.5% of
the injected dose was in the liver. This is substantially more
than the ∼70% of the injected signal that accumulated in the
liver after injection of labeled siRNA in the DPC (Figure 5D).
Furthermore, the kinetics of liver accumulation were different
between the DPCs that had either the siRNA or PBAVE labeled
(Figure 5D,E). As previously noted, the DPC rapidly reached a
plateau by 4 min after injection, while the PBAVE/DPC also
quickly accumulated by 4 min but kept rising for the duration
of the 60 min study (Figure 5D,E).
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Also, in contrast to siRNA-labeled DPC, PBAVE/DPC
underwent minimal renal filtration (Figure 4). The peak
concentration in the kidneys was 9.2 ( 2.4% injected dose/mL
or 5.2 ( 2.8% injected dose at 4 min after injection. It is likely
that this peak corresponds to radioactivity that is in the blood
and not actually being filtered by the kidneys. Only 0.1 ( 0.07%
of the injected dose was present in the bladder at one hour.
Accumulation in Other Organs. The total radioactivity
associated with the liver, kidneys, bladder, heart, spleen, and
lungs accounted for 64% to 100% of the total injected dose for
all samples, suggesting that these were the most important
organs involved in the biodistribution of the labeled molecules
(data not shown). The sum of the activity in these organs was
75.3 ( 11.6% for labeled siRNA, 89.4 ( 6.0% for DPC, and
105.2 ( 16.3% for PBAVE/DPC. Because the %ID per organ
calculations were based on scaled organ volumes, these calcula-
tions contain more error than the %ID/mL, which are derived
from direct measurements. The sum of the %ID per organ being
above 100% can be attributed to this error. In addition, less
than 2% of the injected label from any of the complexes
accumulated in the spleen (data not shown).
CONCLUSION
PET/CT imaging provided a wealth of qualitative and
quantitative information about the biodistribution and kinetics
of the delivery system without the use of lengthy, invasive tissue
distribution studies. One advantage of the in ViVo imaging
method is that it allows one to visualize biodistribution three-
dimensionally throughout the entire body. The DPC delivery
system effectively delivered a significant portion of its conju-
gated siRNA, ∼70% of the total injected dose, to the liver, the
organ of interest. The PBAVE polymer behaved differently from
the siRNA in the DPC in that even more accumulated in the
liver, ∼98% of the injected dose, but at a slower rate.
Approximately 10% of the injected DPC-conjugated siRNA
accumulated in the bladder, but practically none of the PBAVE-
associated signal could be detected there. Further studies are in
progress to understand these differences in targeting of DPC-
associated siRNA and PBAVE.
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strengths, limitations and challenges. Nucl. Med. Commun. 29,
193–207.
ACKNOWLEDGMENT
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L. V., Palmer, L. R., Racie, T., Rohl, I., Seiffert, S., Shanmugam,
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The authors would like to thank Dave Rozema and Darren
Wakefield for helpful discussions, Mark Noble and Alice
Nomura for their help with experiments, and John Floberg for
help with 3D movies. The authors would also like to thank
Christine Wooddell and Hans Herweijer for critically reading
the manuscript.
Supporting Information Available: Three movies depicting
the first 60 min of postinjection dynamics for the 64Cu-labeled

Hybrid PET/CT for Evaluation of Dynamic PolyConjugates
Bioconjugate Chem., Vol. 21, No. 7, 2010 1189
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