Bioconjugate Chem. 1996, 7, 311−316
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Magnetically Labeled Secretin Retains Receptor Affinity to Pancreas Acinar Cells† Tueng T. Shen, Alexei Bogdanov, Jr., Anna Bogdanova, Kirtland Poss, Thomas J. Brady, and Ralph Weissleder* Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts 02114. Received October 12, 1995X
Previously, we have developed a colloidal dextran-stabilized monocrystalline iron oxide nanocompound (MION-46) as a magnetic label for magnetic resonance imaging (MRI). In an effort to use this magnetic label to visualize pancreatic receptor function by MRI in vivo, we investigated the potential of secretin as a vector molecule. Secretin receptors, abundant on exocrine pancreas cells, recognize secretin through its amidated carboxyl terminal. In order to conjugate secretin to MION, we utilized the specific interaction between biotin and streptavidin, since direct conjugation of human secretin to MION has previously resulted in low yields and low affinity of the conjugate (unpublished results). Initially, we biotinylated the N-terminal primary amino group of secretin (60% yield). In a separate step, streptavidin (SA) was immobilized onto the surface dextran molecules of MION (79% yield) by reductive amination. Each secretin molecule was conjugated to one biotin molecule and each MION particle to an average of two SA molecules. The biotinylated secretin was then conjugated to MION through the biotin-streptavidin interaction (90% yield). The secretin-biotin-streptavidin-MION construct thus contained approximately two secretin molecules per MION. An in vitro competitive binding assay of pancreatic acinar cells demonstrated that the magnetically labeled secretin retained affinity to the secretin receptors. In vivo distribution studies in rats showed a significantly higher pancreatic accumulation of the secretin-biotin-streptavidin-MION construct as compared to the control group that had received unmodified MION. Our data indicate that bioactive peptides can be attached to dextran-coated iron oxide particles through the biotin-streptavidin interaction while retaining receptor affinity. Such target-specific agents have potential use in MR imaging to probe for a variety of receptor systems.
INTRODUCTION
Receptor-specific magnetic resonance (MR) contrast agents targeted to the pancreas could improve the accuracy of cancer detection and tumor characterization and facilitate therapeutic drug delivery to the pancreas. Early detection of pancreatic cancer is of prime clinical importance (1). Unfortunately, the sensitivity of tumor detection by conventional imaging methods is relatively low primarily due to the lack of cell-specific contrast agents (2). We have previously developed a dextran-coated (B512F, molecular mass of 11 kDa) monocrystalline iron oxide nanocompound (MION) (3), a stable magnetopharmaceutical which consists of a single crystal core of iron oxide (4.6 ( 1.1 nm) coated with an average of 25 surface dextran molecules (hydrodynamic radius of 20 ( 4 nm). The induced magnetization of MION is 68 emu/g of Fe at 1.5 T, and proton relaxivities are 16.5 (R1) and 34.8 mM-1‚s-1 (R2), respectively (3). MION-46 can be detected in vivo by MR imaging at tissue concentrations as low as 50 nmol of Fe/g of tissue (4), and it has been used as a magnetic label for a variety of MR imaging applications such as imaging of axonal transport (5), ASG receptor function (4), and myocardial infarction imaging (6). When given intravenously in its unmodified form, MION has a blood half-life of 4.5 h in rats and is largely phagocytosed by phagocytic cells in the liver and spleen. † Supported in part by NIH research Grants RO1 CA5964901, 2RO1 CA 54886-OA, and 5T32CA09502-10. * Address correspondence to: Ralph Weissleder, M.D., Ph.D., Department of Radiology, Massachusetts General Hospital, Boston, MA 02114. Phone: (617) 726-8226. Fax: (617) 726-5708. X Abstract published in Advance ACS Abstracts, March 15, 1996.
S1043-1802(96)00003-1 CCC: $12.00
In an effort to direct MION to the pancreatic cells, early feasibility studies utilized cholecystokinin (CCK) as a vector molecule (7). MION-CCK has been shown to be recognized by receptors on exogenous pancreas cells and to decrease the T2 relaxation time of the pancreas (33.8 ( 1.4 ms) but not that of adenocarcinoma (78 ( 6.3 ms). In these early preparations, CCK was chemisorbed onto MION. Although this provides for simple synthesis, the chemisorbed CCK was shown to dissociate from MION. Furthermore, the CCK receptor density on pancreas is not as high as that of other receptor systems, such as secretin receptors (8). In the current study, we investigated the potential of the 27-amino acid peptide secretin as a pancreaticotropic vector molecule. Receptor recognition of this peptide occurs primarily through the amidated carboxyl terminal of secretin which interacts with the receptor present on pancreatic parenchymal cells (9). Previous attempts to covalently conjugate secretin to MION yielded low attachment efficiency (
