Synthesis and In Vivo Fate of Zwitterionic Near‐Infrared Fluorophores

Abstract

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...

Importance of the physicochemical properties of fluorescent dyes for obtaining target-specific in vivo images by membrane-permeable macromolecular imaging probes

“…Furthermore, POH-P, in which HL-P has a larger effective charge moment, exhibited relatively slow renal clearance rate compared with POH-I or POH-A ( Figure 4B). This result compliments those of a recent study in which NIRF dyes with strong surface charges were nonspecifically absorbed in normal tissues [28]. In fact, overall effective charge as measured by ion-exchange chromatography correlates with observations made in vivo.…”

Targeted zwitterionic near infrared fluorescent probe for improved imaging of type 2 cannabinoid receptors

“…28 Previous studies indicate that heptamethine cyanine dyes with an enol ether linkage have significant stability issues, which can be overcome by replacing the enol ether with a C─C linkage. 26,30 In addition, the carboxylate group on ZW760 allows for universal conjugation to various targeting moieties.…”

“…The precursor dye ZW-Cl and the ligand mbc94 were synthesized using the previously reported procedure. 18,28 A Biotage (Charlotte, North Carolina) microwave reactor (model: Initiator þ US/JPN 356007) was employed for the synthesis of the dye ZW760. Flash column chromatography was performed on a Teledyne ISCO (Lincoln, Nebraska) (combiflash RF) purification system with silica gel (standard grade, 60A, Sorbtech, Norcross, Georgia) or C18-reversed phase silica gel (20 to 40 μm, RediSepRf).…”

NIR Fluorescence Imaging Systems with Optical Packaging Technology

“…Since NIR fluorescent light is basically invisible to human eye, specially designed exogenous contrast agents are required to get the NIR fluorescent light within the surgical field to emit photons at the specific locations. When combined with an appropriate NIR fluorophore, these imaging systems enable real-time in vivo imaging to highlight the specific structures desired by the surgeon such as pan and sentinel lymph nodes, 14) peripheral nerves, 15) vasculature, 16,17) bone, 18) pancreas, 19) thyroid and parathyroid glands, 20) as well as many tumorous tissues. 6,21-23) The key to future success will be first-in-human trials of new technology, which in turn requires a comprehensive understanding of such design parameters discussed in this article.…”