Imaging Pancreatic Cancer with a Peptide−Nanoparticle Conjugate

Bioconjugate Chem. 2006, 17, 905−911

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Imaging Pancreatic Cancer with a Peptide-Nanoparticle Conjugate Targeted to Normal Pancreas Xavier Montet, Ralph Weissleder, and Lee Josephson* Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Building 149, 13th Street, Charlestown, Massachusetts 02129. Received February 13, 2006; Revised Manuscript Received April 14, 2006

Designing molecules that bind to targets that become upregulated or overexpressed as normal cells become cancerous is an important strategy for both therapeutic and diagnostic drug design. We hypothesized that pancreatic ductal adenocarcinoma (PDAC) might be imaged with the inverse strategy, that is by the design of a nanoparticleconjugate targeted to bombesin (BN) receptors present on normal acinar cells of the pancreas. Using the fluorescein hapten visualization method to assess the presence of bombesin (BN) receptors, we first demonstrated BN receptors in the normal mouse and human pancreas, but then the lack of BN binding receptors in 13 out of 13 specimens of PDAC. The BN peptide-nanoparticle conjugate, BN-CLIO(Cy5.5), was synthesized and accumulated in the mouse pancreas in receptor dependent fashion, but not in a receptor dependent fashion in other tissues, based on tissue fluorescence measurements. The BN-CLIO(Cy5.5) nanoparticle decreased the T2 of normal pancreas and enhanced the ability to visualize tumor in a model of pancreatic cancer by MRI. The use of BN-CLIO(Cy5.5) nanoparticle as a normal tissue-targeted, T2-reducing contrast agent offers a promising approach to imaging PDAC.

INTRODUCTION Pancreatic ductal adenocarcinoma (PDAC) ranks as the fourth leading cause of cancer death in the US. In 2004, an estimated 32 000 cases will be matched with 31 500 deaths. The mean survival averages 6 months, and less than 5% of all patients survive beyond 5 years (http://seer.cancer.gov/). The dire situation in PDAC relates to the tumor’s intense drug resistance and late diagnosis, among other factors. Late diagnosis is due to the lack of specific symptoms and limitations in diagnostics, allowing the disease to elude detection during its formative stages. Though improved imaging methods are needed to assist in the detection and treatment, few agents have been designed to image molecular targets expressed by PDAC. This is in part due to the fact that many molecularly targeted, radioactive peptides (somatostatin, secretin, bombesin, CCK, VIP) employed in tumor imaging in nonpancreatic organ settings have limited utility as pancreatic imaging agents because of the expression of these receptors on cells of the noncancerous pancreas (16). The large number of receptors present on normal pancreatic stroma, however, suggests an alternative strategy for tumor visualization, one where agents target receptors expressed on normal cells. The strategy of targeting normal rather than tumor cells has worked well with magnetic nanoparticles which selectively decrease the T2 of the normal liver and lymph nodes by targeting macrophages in those tissues and enhance tumor visualization in those tissues by MRI (7, 8). We refer to this approach as a normal tissue-targeted nanoparticle/T2 reduction strategy. We therefore hypothesized that a peptide-nanoparticle conjugate targeted to the bombesin peptide binding receptors on normal pancreatic acinar cells might enhance the visualization of pancreatic tumors in an orthotopic animal model and, potentially, in human PDAC. We noted that the literature suggested a molecular target(s), bombesin peptide binding receptors, was present in the normal pancreas and not PDAC * Corresponding author. Fax: 617-726-5708. Phone: 617-726-5788. E-mail: [email protected].

(9). The distribution of the bombesin binding receptors in PDAC and the normal pancreas is discussed below. Second, bombesin binding receptors internalize ligands, offering a potentially dynamic mechanism of nanoparticle accumulation and amplification (10-12). The broad goal of developing a nanoparticle targeted to normal pancreas for imaging PDAC by MRI included the following subaims: (i) to develop a nanoparticle targeted to bombesin peptide binding receptors in the normal pancreas, (ii) to demonstrate that the nanoparticle decreased the T2 of normal pancreas and, (iii) to demonstrate enhanced tumor/ normal visualization on T2 weighted pancreatic images.

MATERIALS AND METHODS Peptide-Nanoparticle Conjugate Synthesis. Peptides were synthesized by the Tufts Core Facility (Tufts University Core facility, Boston, MA) to obtain a bombesin-like peptide (FITC)BCDDDGQRLGNQWAVGHLM or BN, as well as a scrambled version of the bombesin-like peptide, (FITC)BCDDDGQMLGNHLAVGQWR or ScrBN. Peptides were C-terminal amides. B stands for betalanine and FITC for fluorescein isothiocyanate. The 13 C-terminal amino acids are found in bombesin. Three negatively charged aspartate residues (DDD) were employed as spacers and provide a negative charge bias on the nanoparticle; these increase nanoparticle solubility and decrease nonspecific interactions with negatively charged cells. The amino-CLIO nanoparticle, synthesized as described (13, 14), was first reacted with the N-hydroxysuccinimde ester of Cy5.5 (Amersham Biosciences Corp, Piscataway, NJ) to obtain a ratio of 5.1 dyes per nanoparticle which is denoted CLIO(Cy5.5) (15). The majority of amines remained available for reaction with succinimidyl iodoacetic acid, which then reacted with the sulfhydryl group of the BN or ScrBN peptides (15, 16). The resulting nanoparticles, BN-CLIO(Cy5.5) and ScrBNCLIO(Cy5.5), were identical in composition, but because of the sequence difference, the ScrBN nanoparticle was unable to bind receptors for bombesin. Fluorescein, attached to peptides before conjugation to the nanoparticle, does not appear in this notation; it allows characterization of the peptide:iron ratio based on its absorption at 493 nm. The resulting nanoparticles, BN-

10.1021/bc060035+ CCC: $33.50 © 2006 American Chemical Society Published on Web 05/26/2006

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Figure 1. Binding of bombesin peptide to normal pancreas. (A) Bombesin peptide (BN) or scrambled version (ScrBN) were exposed to normal mouse pancreas. The fluorescein on the peptide is visualized by an anti-fluorescein horseradish peroxidase conjugate. Normal pancreas stained dark purple. BN stains more darkly than ScrBN, indicating specific binding. (B) BN was exposed to a human tissue microarray of normal human pancreas and pancreatic ductal adenocarcinomas. Normal tissue stained more darkly than pancreatic adenocarcinomas.

CLIO(Cy5.5) and ScrBN-CLIO(Cy5.5), had diameters of 35 and 41 nm by laser light scattering, relaxivities of 70 and 62 mM-1 s-1, and 50 and 48 peptides per nanoparticle, respectively. The numbers of dyes or peptides per nanoparticle were based on 8000 iron atoms per amino-CLIO nanoparticle, obtained by the viscosity/light scattering method for estimating nanoparticle core weights (17). The design of the peptide-nanoparticles used here is shown schematically in Figure 1 of Koch (15), except (i) Cy5.5 replaced Cy3.5 as the fluorochrome attached to the dextran, and (ii) peptides coupled were the BN and ScrBN peptides rather than the tat peptide. In both conjugations succinimidyl iodoacetic acid was used to attach the thiol of a cysteine group to the amine of the nanoparticle, so the linker chemistry in the current study is that shown by Koch. To determine the binding of the BN peptide and ScrBN peptides to the normal mouse pancreas, the fluorescein hapten visualization (FHV) and assay (FHA) methods were used, a more complete analysis of which has been published (18). Tissue sections of mouse pancreas, 7 µm thick, were snap frozen. To determine the binding of BN peptide to human tissue specimens, tissue microarrays of pancreatic tumor were obtained (Tristar Technology Group, LLC, Bethesda, MD), which contained 13 cancers and 5 normal pancreas specimens. Tissue microarrays or normal mouse pancreas specimens were incubated for 1 h at room temperature with either BN or ScrBN peptides at 1 µg/ mL diluted in PBS containing 10% rabbit serum, 0.3% sodium azide w/v, and 0.5% H2O2 to block the endogenous peroxidases. Unbound peptide was removed by washing three times with the PBS. An anti-FITC horseradish peroxidase conjugate (Molecular Probes, Eugene OR), diluted into PBS containing

2% human serum to 5 µg/mL, was applied to the sample for 40 min. Unbound conjugate was removed by washing three times with the PBS. Enzyme activity was developed for 8 min using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). For the fluorescein hapten assay, specimens were fixed as above, incubated with ligand, and washed free of unbound ligand as above for the fluorescein hapten receptor visualization method. The immunoassay for immunoreactive fluorescein was employed to quantitate bound fluorescein (19). To the slide chamber was added 500 µL of lysis buffer (PBS, 0.1% BSA, 0.1% Triton X-100, 1 mM 8-anilino-1-naphthalenesulfonic acid with 40 ng/mL of anti-fluorescein-HRP. Incubation was for 2 h at room temperature. Duplicate 200 µL volumes were then transferred to a 96-well plate (MaxiSorp, Nunc) coated with 12.5 ng/mL FITC-labeled BSA (Sigma-Aldrich, St, Louis, MO) and incubated for 1 h at room temperature. Plates were then washed three times (PBS, 0.1% BSA, 0.1% Tween) to remove unreacted anti-fluorescein-HRP, and bound peroxidase was quantitated by absorbance at 650 nm using 200 µL of 3,3′,5,5′tetramethylbenzidine dihydrochloride after 30 min incubation at room temperature. The concentration of fluoresceinated ligand was determined from a standard curve of fluoresceinated ligand diluted in PBS, 0.1% BSA, 0.1% Tween. To determine nanoparticle tissue distribution, normal athymic female nude mice (20-25 g) (n ) 13) were divided in three groups. Group 1 (n ) 3) was injected with PBS and was used to measure the intrinsic fluorescence of each organ. Groups 2 and 3 (n ) 5) were injected with BN-CLIO(Cy5.5) or ScrBNCLIO(Cy5.5) (10 mg Fe/kg), respectively, and sacrificed after 24 h. Tissue fluorescence was obtained using a commercially

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available fluorescence reflectance imaging system (BonSAI system, Siemens, Malvern, PA). Slices of tissues (about 1 mm thick, roughly 5 mm in diameter) were placed on a dark plate. Fluorescence was determined using operator-defined regionsof-interest (ROI) on tissues from both injected and uninjected animals. Background fluorescence was subtracted and the values of BN-CLIO(Cy5.5) and ScrBN-CLIO dependent tissue fluorescence were obtained. To examine whether the BN-CLIO(Cy5.5) could enhance visualization of pancreatic tumors by MRI an orthotopic tumor model was employed. The pancreatic tumor cell line, MIA-PaCa 2 from the American Tissue Culture Collection (ATCC, Manassas, VA), was cultured according to the manufacturer’s instructions. Following anesthesia (Isofluorane, 2%), a paramedian laparotomy allowed access to the pancreas and two million MIA-PaCa2 cells in 10 µL were implanted directly in the pancreatic tail of athymic nude female mice. Histology indicated a single, well-defined tumor per pancreas in all animals with a lack of micrometastases (n ) 5). Mice were imaged on a 4.7T Bruker system (Pharmascan, Karlsruhe, Germany) 6 days after implantation, followed by BN-CLIO(Cy5.5) tail vein injection (10 mgFe/kg), with postcontrast images acquired 24 h later. Isofluorane 2% was used for anesthesia during imaging. Coronal T2* maps were generated using the parameters: TR ) 2000 ms, 16 different TE values from 6.5 to 104 ms, flip angle ) 90°, matrix size ) 128 × 64, number of average ) 4, field of view ) 4.24 × 2.12 cm, slice thickness ) 0.8 mm. Tumor to normal contrast (CNR) ratio was obtained at TR ) 2000 ms, TE ) 52 ms where CNR was taken as the difference in signal intensity of the tumor and normal pancreas divided by the standard deviation of the noise. Hand-drawn regions of interest of clearly hyperintense signal (1-3 mm) were taken as tumor, whose dimensions and position were verified by dissection and histology. After the second MR imaging session, tissues were removed, snap frozen, and cut into 7 µm sections (for hemotoxylin and eosin) or into 15 µm for direct fluorescence microscopy. To characterize the presence of BN binding receptors, the pancreas from athymic female nude mice was removed and digested with collagenase/Dispase enzymes solution (Roche Diagnostics, Penzberg, Germany) for 1 h at 37 °C. Cell suspensions were filtered through strainers of 100 µm (BD Biosciences, Franklin Lakes, NJ) and centrifuged (1000 rpm for 5 min) to obtain disaggregated pancreatic cells. Cells were then incubated with either BN or ScrBN and an anti-acinar cell antibody, obtained from the Developmental Studies Hybridoma Bank developed under the auspice of the NICHD and maintained by the University of Iowa (Department of Biological Sciences, Iowa City, IA) (20). FACS analysis was performed on a FACScalibur (Becton Dickinson, Franklin Lakes, NJ). All animal studies were approved by the MGH animal care committee.

RESULTS Tissue-based radioreceptor assays have found bombesin binding receptors to be absent in human pancreatic ductal adenocarcinomas (9), while studies of GRP receptor mRNA expression in different tissues found bombesin binding receptor mRNA to be abundant in the normal human pancreas (21, 22). (The presence of bombesin binding receptors in the pancreas is discussed more fully below.) To determine if the BN peptide had the desired interaction with normal pancreatic tissue, we used the recently developed fluorescein hapten visualization (FHV) and fluorescein hapten assay (FHA) methods for bound peptide. Here the binding of fluoresceinated peptides to receptors in tissues is visualized or assayed using an anti-fluoresceinhorseradish peroxidase conjugate and horseradish peroxidase substrate (18). An FHV method with normal mouse pancreas

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is shown in Figure 1A, where the tissue was treated with the BN peptide or ScrBN peptides; the former produced a notably darker stain that appeared to be homogeneous. To verify the visual difference in stain intensity from the two peptides, tissues were exposed to either peptide, a volume of lysis buffer was added to dissolve the tissue, and the fluorescein in the lysate was quantified by the fluorescein immunoassay, a procedure we term an FHA method (18). There was 3.5 times more BN peptide than the ScrBN peptide on tissue specimens. The binding of the BN peptide to acinar cells in the mouse pancreas was also evident when disaggregated cells from the pancreas were exposed to the peptide and analyzed by FACS, see Figure 4D. We applied that FHV method to a tissue microarray consisting of 5 normal human pancreas specimens and 13 pancreatic ductal adenocarcinomas. The binding of the BN peptide was assessed using the anti-fluorescein-horseradish peroxidase conjugate. As shown in Figure 1B, the BN peptide produced a notably darker peroxidase stain with normal pancreatic specimens than ductal adenocarcinoma specimens. On the basis of a limited number of specimens, it appears that the BN peptide binds normal human pancreas and not pancreatic adenocarcinomas. To determine the tissue specificity of the BN-CLIO(Cy5.5) nanoparticle, we compared the uptake of the nanoparticle in the pancreas with that of ScrBN-CLIO(Cy5.5), a nanoparticle which has identical physical properties to BN-CLIO(Cy5.5) but which is made with a peptide having a scrambled sequence (Figure 1). As shown in Figure 2A, the pancreas of animals injected with BN-CLIO(Cy5.5) was distinctly brighter than those injected with ScrBN-CLIO(Cy5.5). Organ fluorescence obtained with each nanoparticle, together with tissue autofluorescence, is shown in Figure 2B. The hepatic uptake reflects the fact that nanoparticles are too large for renal elimination, and both BN-CLIO(Cy5.5) and ScrBN-CLIO(Cy5.5) are withdrawn by Kupffer cells of the liver in a nonreceptor dependent fashion. The uptake of the two nanoparticles was similar (p > 0.11-0.57) for all tissues except the pancreas, which had a 3-fold higher fluorescence with BN-CLIO(Cy5.5) than ScrBN-CLIO(Cy5.5), (p < 0.003); see Figure 2C. On the basis of the ratio of fluorescence, receptor-mediated targeting of BN-CLIO(Cy5.5) occurred with the pancreas, with high nonpeptide dependent uptake by the liver and spleen. This method of demonstrating receptor specificity (receptor binding and nonreceptor binding nanoparticles) was employed because the BN peptide has a shorter blood half-life than the BN-CLIO(Cy5.5) nanoparticle and the peptide cannot block receptor-mediated interactions in vivo (Montet and Josephson, unpublished observations). We next examined whether the injection BN-CLIO(Cy5.5) could improve the contrast between normal pancreas and tumor as shown in Figure 3. T2 weighted MR images (TR ) 2000 ms, TE ) 52 ms) before and after the injection of BNCLIO(Cy5.5) of a mouse with a tumor-bearing pancreas are shown in Figure 3A and 3B, respectively. Tumor (arrow) is not visible or barely visible in the precontrast image but after the injection of BN-CLIO(Cy5.5), and darkening of normal pancreas, tumor mass became readily discernible, see Figure 3C (magnified version of 3B). A colorized map of pancreatic T2 values is shown in Figure 3D, superimposed on the T2 weighted image. Tumors were present as well-defined single masses (no micrometastases), whose location was confirmed on dissection, see Figure 3E. With a cutoff of 53 ms for tumor versus normal pancreas, T2 values were 62 ( 6 and 62 ( 5 ms (n ) 5) for tumors, pre- and postinjection, respectively. The average T2 values of the normal pancreas decreased from 46 ( 5 to 39 ( 5 ms (n ) 8) with BN-CLIO(Cy5.5) injection (p ) 0.036). Thus BN-CLIO(Cy5.5) enhanced tumor visualization by selectively decreasing the T2 of the normal pancreas.

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Figure 2. BN peptide targets the BN-CLIO(Cy5.5) nanoparticle to the normal mouse pancreas. (A) Tissue autofluorescence and tissue fluorescence postinjection of ScrBN-CLIO(Cy5.5) or BN-CLIO(Cy5.5) nanoparticle for a normal mouse pancreas. (B) Fluorescence of dissected tissues after injection with nanoparticles and from uninjected animals. (C) Ratio of tissue fluorescence for BN-CLIO(Cy5.5) and ScrBN-CLIO(Cy5.5), obtained after background subtraction. The pancreas was the only organ to preferentially accumulate the BN-CLIO(Cy5.5).

Figure 3. T2 weighted images of pancreas and tumor. Preinjection (A) and postinjection (B) of pancreas and tumor. (C) Image from B at higher magnification. BN-CLIO(Cy5.5) permits tumor (arrows) to be seen. Arrow indicates bulk tumor. (D) Postcontrast agent, colorized T2 map of a pancreas with implanted tumor. Tumor has higher T2s than normal pancreas. Abbreviations: L, liver; P, pancreas; K, kidney; B, bowel. (E) Photograph of partially dissected mouse with pancreas and tumor visible.

We next examined the distribution of BN-CLIO(Cy5.5) within the pancreas by fluorescence microscopy and FACS as shown in Figure 4. Figure 4A and 4B show the tumor margin with H and E stain or fluorescence microscopy, respectively. As shown in Figure 4B, the nanoparticle was preferentially accumulated at the border of the tumor, with substantial regions of low nanoparticle accumulation elsewhere in the micrograph. The BN-CLIO(Cy5.5) nanoparticle was visible in the fluorescein channel, due to fluorescein on the BN peptide or Cy5.5 channel, due to Cy5.5 attached directly to the nanoparticle. Background fluorescence in these channels was corrected using

tissues from noninjected animals. We also injected a mouse, disaggregated the pancreas, and ran the disaggregated cells through a single channel FACS as shown in Figure 4C. Some 8% of the cells were labeled as a distinct population with a median fluorescence of 198 au compared to the median fluorescence of 21 au obtained for the unlabeled cell fraction from the injected animal. We characterized cells from the disaggregated pancreas using a specific anti-acinar cell antibody using dual wavelength FACS as shown in Figure 4D and 4E. Some 33-34% of the cells bound the anti-acinar antibody and these cells also bound the BN peptide and an anti-gastrin

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Figure 4. Accumulation of the BN-CLIO(Cy5.5) nanoparticle in the mouse pancreas. (A) Border (arrows) between tumor and normal pancreas with H and E stain. (B) Fluorescent microscopy of the adjacent tissue slice to that shown in (A). Cy5.5 fluorescence (red) and fluorescein fluorescence (green) are shown. Nanoparticle concentrates at tumor border (arrows). (C) Single channel FACS of disaggregated normal mouse pancreas after injection of BN-CLIO(Cy5.5). Some 8% of cells have Cy5.5 fluorescence. (D) Dual wavelength FACS of cells from disaggregated pancreas binding Cy5 labeled anti-acinar antibody and BN peptide (fluorescein labeled). (E) Cells from disaggregated pancreas binding anti-acinar antibody and anti-GRP. Acinar cells (34% of total pancreatic cells) express GRP receptor and bind BN peptide.

releasing peptide receptor antibody. Since only 8% of the cells were labeled, it appears that most acinar cells (34% of total) cells did not internalize BN-CLIO(Cy5.5).

DISCUSSION A feature of the postinjection BN-CLIO(Cy5.5) images of the mouse pancreas was the heterogeneous signal intensity obtained (Figure 3C). Though we did not correlate local changes in pancreatic signal intensity with higher or lower concentrations of nanoparticles in specific regions of the pancreas, two lines of evidence indicate a generally heterogeneous particle distribution within the pancreas. First, fluorescence microscopy (Figure 4B) showed regions of higher and lower nanoparticle concentration in the nontumor bearing, normal pancreas. Second, FACS analysis of disaggregated acinar cells (Figure 4C, D, E) showed the nanoparticle concentrated in a small percentage of cells. In contrast to these results for the postinjection distribution of BNCLIO(Cy5.5) nanoparticle in the pancreas, examination of dissected pancreatic tissue showed a uniform binding of the BN peptide. First, dual wavelength FACS of cells from the disaggregated pancreas indicated all acinar cells both expressed the GRP receptor (Figure 4E) and bound the BN peptide (Figure 4D). Second, by histochemistry the binding of the BN peptide to the normal mouse pancreas was uniform (Figure 1A). It seems

likely that the heterogeneous change in signal intensity post BN-CLIO(Cy5.5) injection reflects selective regions within the exocrine pancreas which are permeable to BN-CLIO(Cy5.5) and that after extravasating the nanoparticle binds the GRP receptor and is internalized by acinar cells. This hypothesis is supported by the fact that the pancreatic exocrine vasculature is heterogeneous, consisting of both fenestrated and nonfenestrated capillaries (23). We employed a peptide with the 13 C-terminal amino acids of bombesin, see above, which can bind to any of three receptors, the NMB receptor (BB1), the GRP receptor (BB2), or the orphan bombesin binding receptor BB3. These three receptors comprise the bombesin peptide binding receptors in mammals, though they are different gene products with somewhat different ligand specificities. We made no effort to determine whether the NMB, GRP, or BB3 receptors bound the BN peptide or the BN-CLIO(Cy5.5) nanoparticle, noting only that the binding of the BN peptide to pancreatic ductal adenocarcinoma was decreased relative to that seen with normal pancreas and providing a rationale for the development of a BN peptide-nanoparticle conjugate. The distribution of bombesin binding receptors is discussed further below. For the approach we have taken to be useful clinically, receptors binding the BN-CLIO(Cy5.5) nanoparticle must be

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(i) absent in PDAC and (ii) present in the normal pancreas. Absence on PDAC (prior studies): Bombesin binding receptors have been found on dissected cancers from the lung, prostate, and breast, and on many tumor cell lines (5, 24). However, PDAC appears to be different from other cancers; bombesin binding was absent when 29 PDAC specimens were examined using a radiolabeled bombesin peptide (9). Presence on normal pancreas (prior studies): Though the same authors did not detect bombesin binding in the normal human pancreas (9), the mRNA for the GRP receptor is expressed at high levels in the pancreas of primates (21, 22) and the expression of bombesin binding receptors in the rodent pancreas is observed with injected radiolabeled bombesin peptides (4, 12, 25). Absence on PDAC and presence on normal pancreas (our studies): To clarify this situation with respect to the presence of bombesin binding receptors in the normal human pancreas, and verify the observations of Fleischman (9) that receptors are missing in human PDAC, we examined the binding of the BN peptide, first to the normal mouse pancreas (Figure 1A) and then to normal human pancreatic tissue and PDAC specimens. In agreement with Fleischman et al., we could not detect bombesin binding in human PDAC specimens, but were able to detect the binding of our BN peptide in the normal human pancreas (Figure 1B). We therefore conclude that bombesin binding receptors are absent in PDAC and present in the both the normal rodent and normal human pancreas. Difference in results may be due to different detection limits of the methods used, different specificity of peptides or nucleic acid binding probes, or different definitions of what comprises a truly normal, noncancerous specimen of human pancreas. A variety of radioactive bombesin-like peptides have been used to image the overexpression of bombesin binding receptors in animal tumor models (4, 12, 25-28) and clinically (2932). We have taken the inverse targeting approach termed a “normal tissue targeted nanoparticle/T2 reduction strategy”, designing a nanoparticle-based MR contrast agent image targeted to bombesin binding receptors of the normal pancreas. The approach of enhancing tumor visualization by targeting normal tissue takes advantage of the spatial localizing ability of MR, that is its ability to determine the signal from individual voxels, without interference from adjacent voxels. Thus a nanoparticle agent can enhance tumor/normal contrast if it decreases the T2 of normal tissue surrounding a tumor more than the T2 of tumor. Similarly, T2 reductions in other organs such as the liver do affect contrast enhancement in the pancreas. It should be noted that the condition for a normal tissue-targeted nanoparticle, T2 reducing contrast agent is not a complete loss of gene expression/activity in the cancerous state, but only relatively higher activity in a normal tissue than that of tumor. Obviously, the decrease of expression/activity of the marker responsible for selective nanoparticle accumulation in normal tissue should decrease in a highly uniform fashion for a significant class of cancers. Radiolabeled peptides are generally targeted to tumors, and uptake by normal tissue raises background and reduces tumor visualization. In conclusion, we have designed a magneto/fluorescent nanoparticle conjugate that was successfully targeted to the bombesin binding receptors of the rodent pancreas and used it to enhance the visualization of a tumor with a normal tissue targeted nanoparticle/T2 reduction strategy. Heterogeneous changes in the T2 of the normal pancreas were inferred to be due to a heterogeneous vasculature limiting nanoparticle delivery to acinar cells. Preliminary experiments indicate that this heterogeneity can be overcome by using an intraperitoneal mode of administration for BN-CLIO(Cy5.5), which can increase pancreatic targeting of high molecular weight liposomes (33) or chemotherapeutic agents (34). However, even with relatively

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inefficient receptor specific targeting of the pancreas obtained with intravenous administration, the peptide was targeted to the pancreas by tissue fluorescence, reduced the T2 of the normal pancreas, and enhanced the visualization of an implanted tumor.

ACKNOWLEDGMENT Work was supported in part by grants from the Department of Defense (DAMD-170210089) and from the NIH (R01 EB00662, P50 86355, R24 CA92782, and PO1 117969). X.M. was supported by a fellowship from the Swiss National Science foundation (PBGEB-104665).

LITERATURE CITED (1) Korner, M., Hayes, G. M., Rehmann, R., Zimmermann, A., Friess, H., Miller, L. J., and Reubi, J. C. (2005) Secretin receptors in normal and diseased human pancreas: marked reduction of receptor binding in ductal neoplasia. Am. J. Pathol. 167, 959-68. (2) Jensen, R. T., Wank, S. A., Rowley, W. H., Sato, S., and Gardner, J. D. (1989) Interaction of CCK with pancreatic acinar cells. Trends Pharmacol. Sci. 10, 418-23. (3) Reubi, J. C. (2000) In vitro evaluation of VIP/PACAP receptors in healthy and diseased human tissues. Clinical implications. Ann. N. Y. Acad. Sci. 921, 1-25. (4) Nock, B., Nikolopoulou, A., Chiotellis, E., Loudos, G., Maintas, D., Reubi, J. C., and Maina, T. (2003) [99mTc]Demobesin 1, a novel potent bombesin analogue for GRP receptor-targeted tumour imaging. Eur. J. Nucl. Med. Mol. Imaging 30, 247-58. (5) Reubi, J. C. (2003) Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr. ReV. 24, 389-427. (6) Reubi, J. C., and Waser, B. (2003) Concomitant expression of several peptide receptors in neuroendocrine tumours: molecular basis for in vivo multireceptor tumour targeting. Eur. J. Nucl. Med. Mol. Imaging 30, 781-93. (7) Harisinghani, M. G., Barentsz, J., Hahn, P. F., Deserno, W. M., Tabatabaei, S., van de Kaa, C. H., de la Rosette, J., and Weissleder, R. (2003) Noninvasive detection of clinically occult lymph-node metastases in prostate cancer. N. Engl. J. Med. 348, 2491-9. (8) Chachuat, A., and Bonnemain, B. (1995) European clinical experience with Endorem. A new contrast agent for liver MRI in 1000 patients. Radiologe 35, S274-6. (9) Fleischmann, A., Laderach, U., Friess, H., Buechler, M. W., and Reubi, J. C. (2000) Bombesin receptors in distinct tissue compartments of human pancreatic diseases. Lab. InVest. 80, 1807-17. (10) Zachary, I., and Rozengurt, E. (1987) Internalization and degradation of peptides of the bombesin family in Swiss 3T3 cells occurs without ligand-induced receptor down-regulation. EMBO J. 6, 22339. (11) Zhu, W. Y., Goke, B., and Williams, J. A. (1991) Binding, internalization, and processing of bombesin by rat pancreatic acini. Am. J. Physiol. 261, G57-64. (12) Breeman, W. A., De Jong, M., Bernard, B. F., Kwekkeboom, D. J., Srinivasan, A., van der Pluijm, M. E., Hofland, L. J., Visser, T. J., and Krenning, E. P. (1999) Pre-clinical evaluation of [(111)InDTPA-Pro(1), Tyr(4)]bombesin, a new radioligand for bombesinreceptor scintigraphy. Int. J. Cancer 83, 657-63. (13) Josephson, L., Tung, C. H., Moore, A., and Weissleder, R. (1999) High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates. Bioconjugate Chem. 10, 18691. (14) Josephson, L., Perez, J. M., and Weissleder, R. (2001) Magnetic nanosensors for the detection of oligonucleotide sequences. Angew. Chem., Int. Ed. 40, 3204-3206. (15) Koch, A. M., Reynolds, F., Kircher, M. F., Merkle, H. P., Weissleder, R., and Josephson, L. (2003) Uptake and metabolism of a dual fluorochrome Tat-nanoparticle in HeLa cells. Bioconjugate Chem. 14, 1115-21. (16) Reynolds, F., Weissleder, R., and Josephson, L. (2005) Protamine as an efficient membrane-translocating peptide. Bioconjugate Chem. 16, 1240-5.

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Pancreas-Targeted Nanoparticle (17) Reynolds, F., O’Loughlin, T., Weissleder, R., and Josephson, L. (2005) Method of determining nanoparticle core weight. Anal. Chem. 77, 814-7. (18) Montet, X., Yuan, H., Weissleder, R., and Josephson, L. (2006) Enzyme based visualization of receptor-ligand binding in tissues. Lab. InVest. 86, 517-525. (19) Kelly, K. A., Reynolds, F., Weissleder, R., and Josephson, L. (2004) Fluorescein isothiocyanate-hapten immunoassay for determination of peptide-cell interactions. Anal. Biochem. 330, 181-5. (20) De Lisle, R. C., Logsdon, C. D., Hootman, S. R., and Williams, J. A. (1988) Monoclonal antibodies as probes for plasma membrane domains in the exocrine pancreas. J. Histochem. Cytochem. 36, 1043-51. (21) Sano, H., Feighner, S. D., Hreniuk, D. L., Iwaasa, H., Sailer, A. W., Pan, J., Reitman, M. L., Kanatani, A., Howard, A. D., and Tan, C. P. (2004) Characterization of the bombesin-like peptide receptor family in primates. Genomics 84, 139-46. (22) Xiao, D., Wang, J., Hampton, L. L., and Weber, H. C. (2001) The human gastrin-releasing peptide receptor gene structure, its tissue expression and promoter. Gene 264, 95-103. (23) Aharinejad, S., and Bock, P. (1994) Identification of fenestrated capillary segments in microvascular corrosion casts of the rat exocrine pancreas. Scanning 16, 209-14. (24) Zhou, J., Chen, J., Mokotoff, M., and Ball, E. D. (2004) Targeting gastrin-releasing peptide receptors for cancer treatment. Anticancer Drugs 15, 921-7. (25) Karra, S. R., Schibli, R., Gali, H., Katti, K. V., Hoffman, T. J., Higginbotham, C., Sieckman, G. L., and Volkert, W. A. (1999) 99mTc-labeling and in vivo studies of a bombesin analogue with a novel water-soluble dithiadiphosphine-based bifunctional chelating agent. Bioconjugate Chem. 10, 254-60. (26) Smith, C. J., Volkert, W. A., and Hoffman, T. J. (2003) Gastrin releasing peptide (GRP) receptor targeted radiopharmaceuticals: a concise update. Nucl. Med. Biol. 30, 861-8. (27) Rogers, B. E., Bigott, H. M., McCarthy, D. W., Della Manna, D., Kim, J., Sharp, T. L., and Welch, M. J. (2003) MicroPET imaging of a gastrin-releasing peptide receptor-positive tumor in a mouse

model of human prostate cancer using a 64Cu-labeled bombesin analogue. Bioconjugate Chem. 14, 756-63. (28) Lin, K. S., Luu, A., Baidoo, K. E., Hashemzadeh-Gargari, H., Chen, M. K., Brenneman, K., Pili, R., Pomper, M., Carducci, M. A., and Wagner, H. N., Jr. (2005) A new high affinity technetium99m-bombesin analogue with low abdominal accumulation. Bioconjugate Chem. 16, 43-50. (29) Van de Wiele, C., Dumont, F., Vanden Broecke, R., Oosterlinck, W., Cocquyt, V., Serreyn, R., Peers, S., Thornback, J., Slegers, G., and Dierckx, R. A. (2000) Technetium-99m RP527, a GRP analogue for visualisation of GRP receptor-expressing malignancies: a feasibility study. Eur. J. Nucl. Med. 27, 1694-9. (30) Van de Wiele, C., Dumont, F., Dierckx, R. A., Peers, S. H., Thornback, J. R., Slegers, G., and Thierens, H. (2001) Biodistribution and dosimetry of (99m)Tc-RP527, a gastrin-releasing peptide (GRP) agonist for the visualization of GRP receptor-expressing malignancies. J. Nucl. Med. 42, 1722-7. (31) De Vincentis, G., Remediani, S., Varvarigou, A. D., Di Santo, G., Iori, F., Laurenti, C., and Scopinaro, F. (2004) Role of 99mTcbombesin scan in diagnosis and staging of prostate cancer. Cancer Biother. Radiopharm. 19, 81-4. (32) Scopinaro, F., Varvarigou, A., Ussof, W., De Vincentis, G., Archimandritis, S., Evangelatos, G., Corleto, V., Pulcini, A., Capoccetti, F., Remediani, S., and Massa, R. (2002) Breast cancer takes up 99mTc bombesin. A preliminary report. Tumori 88, S258. (33) Syrigos, K. N., Vile, R. G., Peters, A. M., and Harrington, K. J. (2003) Biodistribution and pharmacokinetics of 111In-dTPA-labeled pegylated liposomes after intraperitoneal injection. Acta Oncol. 42, 147-53. (34) Armstrong, D. K., Bundy, B., Wenzel, L., Huang, H. Q., Baergen, R., Lele, S., Copeland, L. J., Walker, J. L., and Burger, R. A. (2006) Intraperitoneal cisplatin and paclitaxel in ovarian cancer. N. Engl. J. Med. 354, 34-43. BC060035+