To address two fundamental and unsolved problems in optical imaging (nonspecific uptake of near‐infrared fluorophores by normal tissues and organs and incomplete elimination of unbound targeted fluorophores from the body), novel zwitterionic near‐infrared fluorophores (e.g., ZW800‐1) were synthesized and their performance compared in vivo to conventional molecules (e.g., ICG) as a function of charge, charge distribution, and hydrophobicity (see picture).

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A longstanding problem in the field of image-guided surgery is the development of ideal near-infrared (NIR) fluorophores. The heptamethine NIR fluorophore indocyanine green (ICG) has been used extensively for image-guided surgery because of its clinical availability and safety. [1][2][3] However, ICG is far from ideal because it exhibits high uptake in the liver, contaminates the gastrointestinal (GI) tract, provides moderate optical properties, [4] is unstable in aqueous media, [3,5] and is unable to conjugate covalently to targeting ligands. [2] Although several classes of novel molecules have been described, [6][7][8][9][10][11][12][13] none to date exhibit simultaneous low background binding, bifunctionality, excellent optical properties, low protein binding, and high serum stability. Although it is intuitive that physicochemical properties, that is, positive/ negative charge density, hydrophilicity/lipophilicity, and charge distribution, will impact in vivo performance, chemical structures that exhibit ideal characteristics have not yet been defined.Previously, our group showed that rigid spherical nanoparticles, such as quantum dots, with a hydrodynamic diameter (HD) 5.5 nm could be rapidly cleared by the kidneys, and exhibit low background binding to normal tissues and organs, but only when the surface charge was neutral, geometrically balanced, and polyionic (referred to herein as zwitterionic for simplicity). [14][15][16][17][18][19] In this study, we explored the hypothesis that NIR fluorescent small molecules would exhibit improved in vitro and in vivo performance if synthesized with zwitterionic charges that are evenly spaced over the molecule to shield the underlying hydrophobicity of the relatively large heptamethine core.We describe two complementary molecules, termed ZW800 AE i where AE i is the charge of the conjugated targeting ligand that will render the final molecule with a net charge of zero (i.e., zwitterionic). ZW800-1 has a net charge = 0 prior to targeting ligand conjugation, and a net charge = 0 after conjugation to a targeting ligand that has a net charge of À1 (that is, a targeting ligand with a net charge of 0 prior to conjugation). ZW800-3a has a net charge = + 2 prior to conjugation, and a net charge = 0 after conjugation to a targeting ligand that has a net charge of À3 (that is, a targeting ligand with a net charge of À2 prior to conjugation). ZW800-1 and ZW800-3a were engineered for high hydrophilicity, with logD (distribution coefficient) at pH 7.4 of À3.56 and À6.95, respectively. Importantly, these molecules have also been engineered with sulfonate groups to impart negative charge and quaternary ammonium cations (quats) to impart positive charge because preliminary results showed that the weaker, more common natural analogues (carboxylic acids and primary amines, respectively) did not exhibit the desired properties.As depicted in Figure 1 a, chloro-substituted NIR fluorophores 8 and 9 were synthesized by employing quats and/or sulfonates (5 or 6) on the indocyanine backbone...

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Biopolymers such as proteins and nucleic acids can be labeled with a fluorescent marker to allow for their detection. Covalent labeling is achieved by the reaction of an appropriately functionalized dye marker with a reactive group on a biomolecule. The recent trend, however, is the use of noncovalent labeling that results from strong hydrophobic and/or ionic interactions between the marker and biomolecule of interest. The main advantage of noncovalent labeling is that it affects the functional activity of the biomolecule to a lesser extent. The applications of luminescent cyanine and squarylium dyes are reviewed.