Fluorescence lifetime imaging can provide valuable diagnostic information relating to the functional status of diseases. In this study, a near-infrared (NIR) dye-labeled hexapeptide (abbreviated Cyp-GRD) was synthesized. In vitro, Cyp-GRD internalized in nonsmall cell lung cancer cells (A549) without observable cytotoxic or proliferative effects to the cells at a concentration up to 1x10(-4) M. Time-domain fluorescence intensity and lifetime imaging of Cyp-GRD injected into A549 tumor-bearing mice revealed that the probe preferentially accumulated in the tumor and the major excretion organs. The fluorescence lifetime of the conjugate at the tumor site was mapped, showing the spatial distribution of the lifetime related to its environment. Additionally, fluorescence intensity image reconstruction obtained by integrating the time-resolved intensities enabled the contrast ratios of tumor-to-kidney or liver in slices at different depths to be displayed. The mean lifetime was 1.03 ns for the tumor and 0.80 ns for the liver when averaging those pixels exhibiting adequate signal-to-noise ratio, showing the tumor had a higher lifetime average and reflecting the altered physiopathology of the tumor. This study clearly demonstrated the feasibility of whole-body NIR fluorescence lifetime imaging for tumor localization and its spatial functional status in living small animals.

Background-Current cancer management faces several challenges, including the occurrence of residual tumor after resection, the use of radioactive materials or high concentrations of blue dyes for sentinel lymph node (SLN) biopsy, and use of bulky systems in surgical suites for image guidance. To overcome these limitations, we developed a real-time intraoperative imaging device that, when combined with near infrared (NIR) fluorescent molecular probes, can aid identification of tumor margins, guide surgical resections, map SLNs, and transfer acquired data wirelessly for remote analysis.

mouse ͉ optical imaging ͉ RGD peptides ͉ tumor ͉ near-infrared A ngiogenesis, the formation of new blood vessels, is the cardinal feature of virtually all malignant tumors (1). Because of this commonality, probing tumor-induced angiogenesis and associated proteins is a viable approach to detect and treat a wide range of cancers. Angiogenesis is stimulated by integrins, a large family of transmembrane proteins that mediate dynamic linkages between extracellular adhesion molecules and the intracellular actin skeleton. Integrins are composed of two different subunits, ␣ and ␤, which are noncovalently bound into ␣␤ complexes (2-4). Particularly, the expression of ␣ v ␤ 3 integrin (ABI) in tumor cells undergoing angiogenesis and on the epithelium of tumor-induced neovasculature alters the interaction of cells with the extracellular matrix, thereby increasing tumorigenicity and invasiveness of cancers (5-9).Numerous studies have shown that ABI and more than seven other heterodimeric integrins recognize proteins and low molecular weight ligands containing RGD (arginine-glycineaspartic acid) motifs in proteins and small peptides (10). Based on structural and bioactivity considerations, cyclic RGD peptide ligands are preferentially used as delivery vehicles for molecular probes for imaging (8,(11)(12)(13) and treating (14-17) ABI-positive tumors and proliferating blood vessels. Until recently, most of the in vivo imaging studies were performed with radiopharmaceuticals because of the high sensitivity and clinical utility of nuclear imaging methods. Particularly, the use of small monoatomic radioisotopes does not generally interfere with the biodistribution and bioactivity of ligands. Despite these advantages, nuclear imaging is currently only performed in specialized centers because of regulatory, production, and handling issues associated with radiopharmaceuticals. Optical imaging is an alternative but complementary method to interrogate molecular processes in vivo and in vitro.Optical imaging for biomedical applications typically relies on activating chromophore systems with low energy radiation between 400 -and 1,500-nm wavelengths and monitoring the propagation of light in deep tissues with a charge-coupled device camera or other point source detectors (18). Molecular optical imaging of diseases with molecular probes is attractive because of the flexibility of altering the detectable spectral properties of the probes, especially in the fluorescence detection mode. The probes can be designed to target cellular and molecular processes at functional physiological concentrations. For deep-tissue imaging, molecular probes that are photoactive in the near-infrared (NIR) instead of visible wavelengths are preferred to minimize background tissue autof luorescence and light attenuation caused by absorption by intrinsic chromophores (19). In contrast to radioisotopes, the NIR antennas are usually large heteroatomic molecules that could impact the biodistribution and activity of conjugated bioactive ligands. However, previous s...

We demonstrate that the structure of carbocyanine dyes, which are commonly used to label small peptides for molecular imaging, and not the bound peptide, controls the rate of extravasation from blood vessels to tissue. By examining several near-infrared (NIR) carbocyanine fluorophores, we demonstrate a quantitative correlation between the binding of a dye to albumin, a model plasma protein, and the rate of extravasation of the probe into tissue. Binding of the dyes was measured by fluorescence quenching of the tryptophans in albumin and was found to be inversely proportional to the rate of extravasation. The rate of extravasation, determined by kurtosis from longitudinal imaging studies using rodent ear models, provided a basis for quantitative measurements. Structure-activity studies aimed at evaluating a representative library of NIR fluorescent cyanine probes showed that hydrophilic dyes with binding constants several orders of magnitude lower than their hydrophobic counterparts have much faster extravasation rate, establishing a foundation for rational probe design. The correlation provides a guideline for dye selection in optical imaging and a method to verify if a certain dye is optimal for a specific molecular imaging application Keywords near-infrared; contrast agent; extravasation; kurtosis; protein bindingThe development of a molecular probe in optical imaging is a complex process that requires substantial and concerted efforts from different disciplines, including chemistry, biology, physics, and engineering. From a chemistry point of view, a typical probe for molecular imaging consists of a targeting moiety, such as peptides, oligosaccharides, small organic molecules and a covalently linked fluorophore. Probes designed for deep tissue and noninvasive optical imaging are different from those for in vitro or cellular studies where desirable features include good fluorescence intensity in the visible range and decent cell

Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst 2 -ANT was determined to have a high affinity for SSTR2. Additionally, 111 In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst 2 -ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64 Cu-CB-4,11-bis(carboxymethyl)-1, 4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst 2 -ANT ( 64 Cu-CB-TE2A-sst 2 -ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors. Methods: Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64 Cu-CB-TE2A-sst 2 -ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64 Cu-CB-TE2A-sst 2 -ANT was compared with that of the 64 Cu-labeled agonist 64 Cu-CB-TE2A-tyrosine 3 -octreotate ( 64 Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and smallanimal PET/CT of 64 Cu-CB-TE2A-sst 2 -ANT was performed. Results: The dissociation constant value for the radiopharmaceutical was determined to be 26 6 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64 Cu-CB-TE2A-sst 2 -ANT showed significantly less internalization than did 64 Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64 Cu-CB-TE2A-sst 2 -ANT from the blood was rapid, whereas the clearance of 64 Cu-CB-TE2A-sst 2 -ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64 Cu-CB-TE2A-sst 2 -ANT than those for 64 Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64 Cu-CB-TE2A-sst 2 -ANT revealed excellent tumor-to-background contrast at 4 h after injection, and standardized uptake values remained high even after 24 h. Conclusion: The PET radiopharmaceutical 64 Cu-CB-TE2A-sst 2 -ANT is an attractive agent, worthy of future study as a PET radiopharmaceutical for the imaging of somatostatin receptorpositive tumors.