MicroPET Imaging of MCF-7 Tumors in Mice via unr mRNA-Targeted Peptide Nucleic Acids Xiankai Sun, †,‡ Huafeng Fang, § Xiaoxu Li, § Raffaella Rossin, † Michael J. Welch, † and John-Stephen Taylor* ,§ Division of Radiological Sciences, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110, and Department of Chemistry, Washington University, St. Louis Missouri 63130. Received September 14, 2004; Revised Manuscript Received February 3, 2005 As more becomes known about the expression profiles of normal and cancerous cells, it should become possible to design antisense-based imaging agents for the early detection of cancer noninvasively. In this report, we rationally designed and synthesized three antisense and one sense hybrid PNA (peptide nucleic acid) to the unr mRNA that is highly overexpressed in a breast cancer cell line (MCF-7). The conjugates had a four-lysine tail at the carboxy terminus for cell permeation and a DOTA (1,4,7,10- tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid) chelating moiety at the amino terminal end for chelating 64 Cu for biodistribution and microPET imaging studies. Biodistribution of two 64 Cu-labeled conjugates with antisense and sense sequences (PNA50 and PNA50S) showed high uptake and long retention in kidney and low uptake and efficient clearance in blood and muscle in normal balb/c mice when administered intravenously or intraperitoneally. Intraperitoneal administration, however, gave a much slower release rate. MCF-7 tumors (100-320 mg) in CB-17 SCID mice were imaged with all four 64 Cu-labeled PNA conjugates by microPET, but the image contrast varied with different time points and different conjugates. Of the conjugates studied, 64 Cu-DOTA-Y-PNA50-K4 showed the best tumor image quality at all time points with a tumor/muscle ratio of 6.6 ( 1.1 at 24 h postinjection, which is among the highest reported for radiolabeled oligonucleotides. Our work further strengthens the potential of antigene and antisense PNAs to be utilized as specific molecular probes for early detection of cancer and ultimately for patient specific radiotherapy. INTRODUCTION Noninvasive imaging techniques are revolutionizing the way of understanding diseases at the cellular and molecular levels. Among the current available imaging modalities, positron emission tomography (PET) has demonstrated its great potential in the field of molecular imaging. The success of PET is due to its superior sensitivity and specificity in diverse applications and the ability to quantitatively analyze the regions of interest (1, 2). Since the completion of human genome sequence, there has been considerable research interest in assessing gene expression through noninvasive molecular imaging approaches. Currently, the number of genes is estimated to be between 24 000 and 30 000 and that alternative polyadenylation and splicing could result in the formation of between 46 000 and 85 000 messenger RNAs (mRNA) (3). Of the various PET probes that have been developed to image gene expression in small animal models, oligo- deoxynucleotides (ODNs) appear to be an inexhaustible gold mine for the development of new tracers with high specificity considering that an ODN with more than 12 bases could target a unique sequence in the whole human genome (4). Naturally occurring ODNs cannot be directly used for nuclear imaging because they are rapidly degraded in vivo by endo- and exonucleases (5). Furthermore, ODNs can cause degradation of the target mRNA by RNAse H. To increase the in vivo stability of ODNs without significant alteration of their pharmacokinetics and targeting properties, many chemical modifications have been made to the sugar-phosphate backbone. Modifica- tions include morpholino, phosphorothioate, phosphoro- amidate, methylphosphonate, 2′- or 3′-modifications, 2′- 4′ bridges (locked nucleic acid), and complete replacement of the backbone with an amide backbone (peptide nucleic acid or PNA) (6-8). PNAs are unique types of oligonucleo- tides, which were initially introduced by Nielsen et al. in 1991 as ligands for the double-stranded DNA recogni- tion (9). They are synthetic DNA mimics featuring a chain with repeating N-(2-aminoethyl) glycine units instead of the sugar-phosphate backbone. Because of the structural characteristics (e.g., neutral and flexible), PNAs are resistant to in vivo enzymatic degradation and bind complementary DNAs or RNAs with high affinity and specificity even under low ionic strength (10). They also do not activate RNAse H degradation of mRNA. Recent work has shown that PNAs can be used as molecular hybridization probes (11), nuclear imaging tracers (8, 12-16), and to control gene expression and splicing (6, 17-19). Using antisense PNAs as molecular imaging probes has a major obstacle in that they are not able to penetrate biologic membranes. To overcome this obstacle, research- ers have resorted to drug delivery techniques such as * To whom correspondence should be addressed. Tel: 314- 935-6721. Fax: 314-935-4481. E-mail: taylor@wustl.edu. † Washington University School of Medicine. ‡ Current address: Department of Radiology, the University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390. § Washington University. 294 Bioconjugate Chem. 2005, 16, 294-305 10.1021/bc049783u CCC: $30.25 © 2005 American Chemical Society Published on Web 02/26/2005