Doubly Strapped Zwitterionic NIR-I and NIR-II Heptamethine Cyanine Dyes for Bioconjugation and Fluorescence Imaging.

Heptamethine cyanine dyes enable deep tissue fluorescence imaging in the near infrared (NIR) window. Small molecule conjugates of the benchmark dye ZW800-1 have been tested in humans. However, long-term imaging protocols using ZW800-1 conjugates are limited by their instability, primarily because the chemically labile C4'-O-aryl linker is susceptible to cleavage by biological nucleophiles. Here, we report a modular synthetic method that produces novel doubly strapped zwitterionic heptamethine cyanine dyes, including a structural analogue of ZW800-1, with greatly enhanced dye stability. NIR-I and NIR-II versions of these doubly strapped dyes can be conjugated to proteins, including monoclonal antibodies, without causing undesired fluorophore degradation or dye stacking on the protein surface. The fluorescent antibody conjugates show excellent tumor-targeting specificity in a xenograft mouse tumor model. The enhanced stability provided by doubly strapped molecular design will enable new classes of in vivo NIR fluorescence imaging experiments with possible translation to humans.

Cytotoxicities of Tumor-Seeking Dyes: Impact on Future Clinical Trials.

Heptamethine (Cy7) dyes with meso-Cl substituents injected intravenously (iv) into mice accumulate in tumors and persist there over several days. We believe this occurs via meso-Cl displacement by the only free cysteine residues of albumin; therefore, conjugating tumor-seeking dyes with fragments can increase selective therapeutic delivery to tumors and drug residence. This strategy has elevated significance recently because the first tumor-seeking dye-drug conjugate has moved into clinical trials. Options for further clinical research include modifying the dye, and use of preformed albumin adducts instead of dyes alone. Herein we show correlations of cytotoxicities, lipophilicities, organelle localization, apoptosis, cell-cycle arrest, wound healing/migration assays, and reactivities/affinities with human serum albumin are difficult to observe. However, our studies arrived at an important conclusion: preformed dye-drug-HSA adducts are less cytotoxic, and therefore preferable for subsequent clinical work, relative to direct injection of meso-Cl-containing forms.