ZW800-1, a zwitterionic near-infrared fluorophore, and its cyclic RGD peptide derivative cyclo-(RGDyK)-ZW800-1

Indocyanine green (ICG) is a noninvasive near-infrared (NIR) fluorescence (NIRF) imaging dye that is approved by the United States Food and Drug Administration (FDA) for ophthalmic angiography to determine cardiac output and liver blood flow and function. This dye is also used in cancer patients to map sentinel lymph nodes (SLN), for the detection of solid tumors, and for angiography during reconstructive surgery (2). Methylene blue (MB) is another NIRF dye that has been approved by the FDA for the treatment of drug-induced methemoglobinemia , but MB is also used to visualize SLNs with ultrasound imaging in the clinic (3). Two other commercially available NIRF dyes, Cy5.5 and IRDye800-CW (CW800), have been coupled with peptides or antibodies and successfully used for the targeted visualization of neoplastic tumors in rodents (4). Although the NIRF dyes exhibit low autofluorescence, tissue absorbance, and scatter at NIR wavelengths (700–900 nm), a major limitation of using these dyes in the clinic is that they often show high nonspecific binding and have high tissue uptake and retention (5). In addition, these dyes are cleared from circulation primarily through the hepatobiliary pathway and generate high background signals in the gastrointestinal tract, which results in the masking of signals from cancerous lesions that may be present in tissues located around the digestive system (5). The high background signals interfere with distinguishing the tumors from normal tissues during NIRF-guided resection of the lesions (6). Physiochemical properties such as positive/negative charge density, charge distribution, and hydrophilicity/hydrophobicity (or lipophilicity) are known to affect the protein binding, serum stability, and in vivo biodistribution characteristics of compounds and imaging agents, including probes of the NIRF variety (7). In this regard, MB carries a small net positive charge (+1), whereas ICG, Cy5.5, and CW800 carry a net negative charge (−1, −4, and −4, respectively) and are hydrophobic (Table 1) (6). The physiochemical properties of these NIRF dyes are discussed in detail by Gioux et al. (6).Results obtained from a biodistribution study of NIRF nanoparticles (NPs) with different chemical compositions, hydrodynamic diameters, shapes, and surface charges administered to rats through the pulmonary route showed that zwitterionic NPs (i.e., NPs with a net zero charge or charge-neutral), compared with cationic or anionic charged NPs, were rapidly absorbed from the lungs and were moved to the SLN and into blood circulation for clearance through the kidneys (8). Similar results were reported with zwitterionic quantum dots (QDs) conjugated to CW800 and decorated with a limited number of small molecule (2-(3-amino-3-carboxypropyl)) or peptide (cyclo-RGD-yK) ligands that targeted the prostate-specific membrane antigen and the αvβ3 integrin receptor, respectively (9). On the basis of these studies, it was hypothesized that NIRF agents with a net zwitterionic charge would probably behave in a manner similar to the zwitterionic NPs and QDs under in vivo conditions (7). In addition, it was expected that the charge-neutral NIRF agents would generate superior images compared with NIRF probes that carried a net anionic or cationic charge. To test this hypothesis, the biodistribution of a series of heptamethine indocyanine NIRF probes with varying net charges, e.g., ICG (net charge −1), CW800 (net charge −4), RS800 (a derivative of CW800, net charge −2), ZW800-1 (a derivative of CW800, net charge 0), and ZW800-3a (a derivative of CW800, net charge +2) was investigated in mice (7). In another study, cyclo-(RGDyK) conjugated to ZW800-1 (cRGD-ZW800-1) was compared with cyclo-(RGDyK) conjugated to CW800 (cRGD-CW800) and cyclo-(RGDyK) conjugated to Cy5.5 (cRGD-Cy5.5) for the visualization of xenograft tumors that overexpressed integrin αvβ3 receptors in mice (5). In addition, fibrinogen (FBG) labeled with the NIRF dyes (FBG-ZW800, FBG-CW800, and FBG-Cy5.5, respectively) was evaluated for the detection of fibrinogen-positive thrombi (blood clots) in mice (5).