Targeted fluorophore-labeled contrast agents are moving toward translation to human surgical use. To prepare for
future clinical use, we examined the performance of potential ligands targeting the epidermal growth factor receptor,
α5β3 integrins, and GLUT transporters for their suitability as directed contrast agents. Each agent was labeled with
IRDye 800CW, and near-infrared dye with excitation/emission wavelengths of 789/805 nm, which we determined
had favorable toxicity characteristics. The probe molecules examined consisted of Affibodies, nanobodies, peptides,
and the sugar 2-deoxy-D-glucose. Each probe was tested for specific and non-specific binding in cell based assays.
All probe types showed good performance in mouse models for detecting either spontaneous tumors or tumor
xenografts in vivo. Each of the probes tested show promise for future human clinical studies.
Fluorophore-labeled contrast imaging agents are moving toward clinical use as aids in nodal staging and intraoperative
resection of tumors. Near-infrared fluorophores with defined toxicity properties will be needed before these agents can
be translated to the clinic. The near-infrared dye IRDye 800CW is frequently used in its N-hydroxysuccinamide (NHS)
ester form for labeling these agents. Following conjugation or breakdown of a labeled ligand, excess NHS ester is
converted to the carboxylate form. We report here the results of a preliminary toxicity study on IRDye 800CW
carboxylate in preparation for its use as a labeling moiety for targeted contrast agents. Male and female Sprague Dawley
rats were given a single intravenous or intradermal administration of IRDye 800CW carboxylate; indocyanine green
was used as a comparative control. Following administration of varying doses of either the dyes or saline, animals were
observed for up to fourteen days during which time, hematological, clinical chemistry, enzymological, and histological
testing was performed on animal subgroups. Under the conditions tested, a single administration of IRDye 800CW
carboxylate intravenously at dose levels of 1, 5 and 20 mg/kg or 20 mg/kg intradermally produced no pathological
evidence of toxicity. A dose of 20 mg/kg was identified as the NOAEL (no observed adverse effect level) following IV
or ID routes of administration of IRDye 800CW.
The study of disease processes requires a number of tools for detection of proteins and biomarkers in cell and animal
based assays. Near infrared (NIR) technologies offer the advantage of high signal without interference from
background producing factors such as tissues, blood, or plastics. NIR fluorescence quenching biochemical assays
employing a novel NIR quencher are homogeneous and sensitive. NIR-based immunocytochemical assays offer a
means of quantitatively evaluating cell signaling pathways. The technology can be extended to the development of
targeted molecular imaging agents for disease analysis in animal models. We describe here model assays for each of
these categories. A fluorescence quenching caspase-3 assay was developed employing a novel, broadly applicable
quencher dye suitable for use with both visible and NIR dye chemistries. An NIR cell based assay is described for
assessment of phosphorylation of p53 in response to a cellular stimulus. Finally, we describe the development and
application of a targeted NIR optical imaging agent for monitoring tumor growth in whole animals. The NIR
biochemical and cell based assays are robust with Z' factors greater than 0.7. The use of an IRDye <sup>(R)</sup>800CW-labeled
cyclic RGD peptide is presented as a model for development and application of targeted imaging agents. NIR
technologies are compatible with the complete spectrum of assay needs for disease analysis and therapeutic
Optical imaging is a rapidly developing field of research aimed at non-invasively interrogating animals for disease
progression, determining the effects of a drug on a particular pathology, assessing the pharmacokinetic behavior of a
drug, or identifying molecular biomarkers of disease. One of the key components of molecular imaging is the
development of specific, targeted imaging contrast agents to assess these biological processes. The development of
robust fluorochrome-labeled optical agents is a process that is often underestimated in terms of its complexity. We
describe here the development process and performance issues for three different optical agents: IRDye 800CW EGF
(epidermal growth factor), IRDye<sup>(R)</sup> 800CW 2-DG (2-deoxy D-glucose), and an IRDye 680 BoneTag<sup>TM</sup>. In vitro
competitive assays were developed for two of the markers to demonstrate specificity. Specificity was confirmed in
animal studies. Uptake of IRDye 800CW 2-DG was also examined by near-infrared confocal microscopy. Histological
examinations were performed on target and non-target tissues following the completion of the imaging studies. The
issues unique to the development of each labeled marker are discussed.
The use of fluorescently labeled probes for membrane-based analysis of proteins and nucleic acids have been unsuccessful due to high background fluorescence of the membranes. We have developed a system for the analysis of proteins and nucleic acids using near infrared (IR) fluorescent dyes and a new scanning instrument, the Odyssey Infrared Imager. Nucleic acid probes can be directly labeled with carbodiimidederivatized IR dyes in a 10 minute labeling and clean up procedure. In a Northern blot format, mRNA from genes such as mouse 3-actin, mouse GAPDH, and cyclophilin mRNA have been detected in as little as 0.1 tg of total RNA. In immunoblotting assays, JR detection of proteins was as sensitive as chemiluminescence and routinely enabled detection of 1-10 pg of protein. Signal transduction events in the MAP kinase pathway were analyzed using two-color immunoblotting. Phosphorylated and non-phosphorylated proteins were detected and quantitated simultaneously without the need for stripping and reprobing. A model system for assessing protein-nucleic acid interactions by EMSA was developed based on the binding of T7 RNA polymerase to its promoter. Varying amounts of protein were combined with a PCR-generated IRD800labeled DNA fragment. Protein binding could be monitored quantitatively following scanning of the gel on the Odyssey Infrared Imager. The Odyssey Infrared Imaging system permits two-color analysis of proteins and nucleic acids with very high signal to noise ratios.
Near infrared (NTR)fluorescentacycloterniinators were tested as substrates in the Sanger enzymatic method ofDNA sequencing. The acyclic triphosphates ofadenosine, uridine, guanosine, and cytidine (AcyNTP) were labeled with a heptamethine carbocyanine dye via a propargylamino linker to the purine or pyrimidine base. Dye-labeled AcyNTPs which are lacking in the sugar moiety positions equivalent to the C-2 and C-3 ofthe ribose functioned similarly to chain-terminating dideoxynucleotides (ddNTPs). These fluorescent nonnucleotide analogs were incorporated by a mutant, thermostable Taq DNA polymerase with the same efficacy and fidelity as traditional ddNTPs. Sequence read length and basecalling accuracy were comparable for both dye-acyclotenninator and dye-primer sequencing methods. In two primer walking projects, cycle sequencing with fluorescent AcyNTPs achieved a mean sequence read length of 1,090 bases with 99. 1% accuracy at one kilobase read length. The cyanine dye-labeled acycloterminators produced electropherograms in which weak T peaks follow G peaks. In cases of polymorphism, such peak height variability may make it difficult to distinguish the presence or absence of a heterozygote at a specific site.