CT for Noninvasive Pharmacokinetic Evaluation of

Jun 16, 2010 - Corresponding Author: Vladimir S. Trubetskoy, Address: 465 Science Dr., Madison, WI 53711. E-mail: [email protected], Telep...
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Bioconjugate Chem. 2010, 21, 1183–1189

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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 64 Cu 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.

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 polymers 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: [email protected], Telephone number: (608)316-3925, Fax number: (608)441-0741. † Department of Pharmaceutical Sciences, University of Wisconsin Madison. ‡ Roche Madison Inc. § Department of Radiology, University of Wisconsin - Madison.

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 independently 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 additional 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 microscopy 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. 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 microPET/CT hybrid imaging with 64Cu DOTA-labeled DPCs. The half-life of 64Cu is 12.7 h, which is reasonable for imaging

10.1021/bc900558z  2010 American Chemical Society Published on Web 06/16/2010

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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-NHSester 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 (DOTANM). In preparation of precursor 4 we utilized reductive

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alkylation of amine 3 with 4-[(N-(2-chloroethyl)-N-methyl]aminobenzaldehyde 2 followed by Boc-deprotection with CF3CO2H (Figure 1) (14, 15). Aldehyde 2 was prepared from 4-[N-(2hydroxyethyl)-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 1 H NMR. N-(3-aminopropyl)-N-[3-[[[4-[(2-chloroethyl)methylamino]phenyl]methyl]amino]propyl]-N,N-dimethylammonium tetrakis(trifluoroacetate) 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 DOTAPBAVE 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 modified 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. 64 Cu 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 DOTAsiRNA 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 studies 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 performed in accordance with all guidelines and were approved

Hybrid PET/CT for Evaluation of Dynamic PolyConjugates

Figure 2. Schematic representations of (A) Labeled 64Cu (64Cu), (B) 64 Cu 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

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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. 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 64 Cu 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.

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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 64 Cu 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 postinjection 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). 64 Cu-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

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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. In comparison to free 64Cu, more of the labeled siRNAassociated radioactivity appeared in the kidneys, especially at the early time points (Figure 3B,D,F). The maximum labeled

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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). 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. 64 Cu-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

(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

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in hepatocytes (3). This is consistent with receptor ligand mediated uptake of DPC. 64 Cu-Labeled PBAVE/DPC (PBAVE/DPC) Imaging Studies. The DPC contains an excess of the NAG-targeted PBAVE not conjugated with siRNA. Thus, the tissue distribution 64Culabeled 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). 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 calculations 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 threedimensionally throughout the entire body. The DPC delivery system effectively delivered a significant portion of its conjugated 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 PBAVEassociated signal could be detected there. Further studies are in progress to understand these differences in targeting of DPCassociated siRNA and PBAVE.

ACKNOWLEDGMENT 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

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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.

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