Superior Near-IR Dyes for Bioimaging

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Optical imaging is an advanced technique to image biotechnology products like gels, sensors, and micro arrays. It is also used increasingly to monitor specific molecular pathways in animals. This latter technique uses fluorescent dyes that absorb and emit near-infrared (NIR) radiation (650-900 nm) a spectral window where hemoglobin and water absorb minimally and therefore allow photons to penetrate several centimeters through tissue. This proposal builds on our recent discovery that squaraine rotaxanes (SRs) are among the world's brightest and most stable fluorescent NIR dyes. However due to their poor solubility under aqueous conditions, their usefulness in biological applications remains a challenge thus limiting their commercial appeal. Our plan is therefore to improve dye performance by introducing water solublizing sulfonate or phosphonic acid groups into the molecular structure. With further development, they are likely to become superior replacements for the popular cyanine (Cy5) dyes and become extremely useful probes for both in vitro and in vivo imaging applications. The overall goal for Phase I is to prepare novel water soluble SR dyes and compare the properties of various bioconjugates that have been labeled with SR or Cy5. The two Specific Aims of the present proposal are: 1) SA1 to be performed at MTTI - Synthesize novel water soluble SR NHS esters incorporating multiple sulfonate or phosphonic acid groups and conjugate to 3 different types of biomolecules, an antibody, a protein and a vitamin for comparison with Cy5 analogues. The first goal is to produce water soluble squaraine rotaxane probes having sulfonate or phosphonic acid groups. This will be followed by conjugation of the squaraine rotaxane probes as well as Cy5 as control probe to IgG protein, streptavidin and a folic acid derivative. 2) SA2 to be perfomed at UND - In vitro and in vivo evaluation of bioconjugates. In vitro molecular recognition performance of the SR and Cy5 fluorescent IgG and streptavidin bioconjugates developed at MTTI will be characterized using standard gel electrophoresis and streptavidin/biotin protocols. In vitro and In vivo molecular recognition performance of fluorescent SR and Cy5 folate conjugates will be determined using cultured cells that express high levels of the folate receptor and in whole animal imaging studies of murine xenograft tumor models. The goal is to demonstrate the advantage of using molecular probes labeled with SR dyes in 3 different systems compared with Cy dyes. PA-08-050