Colton IJ, Carbeck JD, Rao J, Whitesides GM. highly anionic surface charge. This chemical and geometric configuration Longdaysin results in relatively high nonspecific binding and use, are cleared by renal filtration so as to prevent contamination of the gastrointestinal tract. The hypothesis guiding our work is that targeted zwitterionic molecules, molecules containing evenly spaced and interspersed positive and negative charges, might recapitulate the results seen previously with targeted zwitterionic nanoparticles15C18. In that prior work, zwitterionic surface coatings, but not anionic or cationic coatings, resulted in much lower than expected nonspecific uptake. In a previous study characterizing the properties of NIR fluorophores, we found that the zwitterionic heptamethine indocyanine NIR fluorophore ZW800-1 exhibits low serum binding, ultralow nonspecific tissue background and rapid elimination from the body via renal filtration19. Moreover, ZW800-1 has notable optical properties including high extinction coefficient and Longdaysin quantum yield, ensuring maintenance of a high signal. Here we conjugated the zwitterionic NIR fluorophore ZW800-1 to various targeting ligands and compared these with conjugates made using CW800 and Cy5.5, in a broad spectrum of biomedical optical imaging assays. CW800 and Cy5.5 were chosen for comparison as they have the best reported performance and highest number of citations in the literature to date. Our results suggest that targeted zwitterionic NIR fluorophores outperform conventional NIR fluorophores in a wide variety of and assays. RESULTS Physiochemical properties of targeted NIR fluorophores To create targeted ZW800-1 (emission 800 nm), CW800 (heptamethine indocyanine with a cyclohexane center that emits at 800 nm) and Cy5.5 (linear pentamethine that emits at 700 nm), we covalently conjugated each fluorophore to a cyclic peptide consisting of Arg-Gly-Asp-D-Tyr-Lys (cRGD), which specifically binds integrin v3 (Fig. 1a). ZW800-1 was synthesized using a process compatible with good manufacturing practices (Supplementary Fig. 1)20. Because of the starting anionic charge, cRGD-CW800 and cRGD-Cy5.5 exhibited net surface charges of ?4 and a highly Longdaysin unbalanced (i.e., dipole-like) charge-to-hydrophobicity distribution over their surfaces. In contrast, cRGD-ZW800-1 was very hydrophilic (logD = ?9.77), had a net surface charge of 0 and had an extremely well-balanced charge distribution over its surface (Fig. 1a). Molecular modeling (Fig. 1a) suggested that the indole nitrogen may be buried after conjugation and thus would not make a notable contribution to surface charge. Open in a separate window Open in a separate window Open in a separate window Figure 1 Targeted NIR fluorophores and improved SBR during cell-based assays(a) Chemical structure, molecular weight (MW), logD, net surface charge and 3D modeling of the geometrical position of charge and hydrophobicity of cRGD-ZW800-1, cRGD-CW800 and cRGD-Cy5.5 at pH 7.4. Red = negative charge; Blue = positive charge; Gray = hydrophobicity. (b) Live cell binding assay for cRGD-ZW800-1, cRGD-CW800 and cRGD-Cy5.5 (top 2 rows) or NIR fluorophores alone (bottom row) in M21 (v3-positive) and M21-L (v3-negative) melanoma cell lines after incubation with 2 M of each molecule for 30 min at 37 C. Scale bars = 100 Ly6a m. All NIR fluorescence images have identical exposure times and are normalized to peak signal. (c) Antibody binding assay for secondary antibodies conjugated with ZW800-1, CW800 and Cy5.5 in MDA-MB-361 (Her2-positive) and MDA-MB-231 (Her2-negative) human breast cancer cell lines in the presence (top 2 rows) or absence (bottom 2 rows) of the anti-Her2 primary antibody. Scale bars = 50 m. All NIR fluorescence images have identical exposure times and are normalized to peak signal. Cell binding assays, histology and immunoblotting We next confirmed the specificity of targeted NIR fluorescent ligands to.