Avidin conjugated to tetramethyl-6-carboxyrhodamine-QSY7

Early detection of cancer helps in the development of a proper treatment and monitoring regimen that may result in a suitable prognosis for the patient (1-3). Although invasive methods are often used for the detection of cancer, they have limitations because these procedures may detect the neoplasm only in a specific location and cannot determine whether the cancer has metastasized to other parts of the body. Also, the various imaging techniques and modalities available for the non-invasive detection of cancer have low sensitivity and/or resolution that are insufficient to detect and quantify microscopic tumors or a small cluster of cancerous cells (4). In addition, some of the contrast agents used for imaging purposes are known to have toxic side effects (5). An attractive alternative to radionuclides or contrast agents is the use of fluorescent optical imaging agents that can be used to visualize and manage or treat clinical pathology, including that of cancer (6-9). However, fluorescent dyes used for imaging can be toxic or nonspecific, and the signal generated by these agents may be masked by autofluorescence, leading to a low signal/background ratio (10). Also, the attenuation of the fluorescent signal in the tissue is such that only superficial tumors can be detected in humans. Investigators have proposed the use of targeted fluorescent probes as a solution to improve the signal/background ratio with the optical imaging agents, and they have shown that fluorescence dye conjugated to avidin can be used to detect submillimeter disseminated peritoneal tumors under and conditions only if direct access to the peritoneal tumor is possible (11, 12). In addition to binding to biotin, avidin (a 68-kD tetrameric glycoprotein) is also known to bind to the D-galactose receptor, which belongs to the lectin class of molecules (proteins that can bind to the carbohydrate groups of glycoproteins and glycolipids) and has been reported to be expressed on the surface of ovarian, gastric, colon, and pancreatic cancer tumor cells (12, 13). Because of its specificity to bind to cell-surface ligands, the use of avidin-therapeutic drug conjugates as a method to deliver and facilitate the uptake of anticancer drugs by cancer cells was also suggested to treat this ailment (14). The net positive charge on avidin facilitates its binding to lectins or similar molecules that are negatively charged at physiological pH (11). Lectin-bound avidin or its conjugates are rapidly internalized by the cell, and the unbound avidin and its conjugates are quickly cleared from circulation through the liver. Therefore, this phenomenon helps generate very high target signal/background ratios in small animals (11). Ogawa et al. proposed the use of fluorophores that could be activated only after binding to the target to further improve the target/background ratios obtained with the optical imaging agents (15). These investigators developed a targeted and activatable fluorophore-quencher (FQ) probe that could be used for the optical imaging of tumors. In the native state the signal from the fluorophore is quenched by a quencher molecule. A fluorescence signal would be obtained from the fluorophore only after the FQ probe was bound to, internalized, and activated by the target cells. The mechanism of quenching and generating fluorescence from the FQ probe is discussed elsewhere (16). Ogawa et al. investigated the use of tetramethyl-6-carboxyrhodamine (TAMRA, fluorophore)-QSY7 (quencher) pair conjugated to avidin (Av-TM-Q7) for the detection of cancer cells under conditions and also in mice bearing SHIN3 cell line (of human ovarian cancer origin; these cells have a surface expression of the D-galactose receptor) tumors (15).