Fluorescence Imaging Using Long-Wavelength Cyanine Dyes and Targeted Molecular Probes

Optical imaging and diagnostic applications employing fluorescence dyes and molecular probes is having increased impact in both research and clinical settings. The rapid evolution of instrument manufacturing, including advancements in cameras, light sources, and multi-modal imaging platforms, has markedly improved the sensitivity and quality of fluorescence-based methods. Furthermore, fluorescence-based techniques offer a non-invasive and cost-effective alternative to many conventional imaging modalities. Thus, there is considerable value in developing novel fluorescent dyes and molecular probes that are endowed with superior chemical and photophysical properties. This thesis investigates the molecular dynamics and biological targeting mechanisms of deep-red and near-infrared cyanine dyes, as well as the design of affinitybased molecular probes for imaging and diagnostic applications. Chapter 1 summarizes common strategies observed in nature for anchoring membrane proteins, which serve as a blueprint for the design of synthetic dyes and three-component molecular probes that are suitable for fluorescent labeling of cells or liposome membranes. In Chapter 2, spontaneous transfer of the near-infrared dye Indocyanine Green (ICG) from liposomes to bovine serum albumin proteins located in the external solution is characterized through Förster Resonance Energy Transfer (FRET)-based experiments. ICG, a clinically approved heptamethine cyanine dye with favorable near-infrared absorption and fluorescence profiles, is used often in fluorescence imaging and phototherapeutics. A systematic study of lipophilic additives determined that the presence of clinically approved antioxidant a-tocopherol greatly enhanced ICG retention within the liposome membrane. Chapter 3 introduces a deep-red complementary analogue of ICG, termed Indocyanine Blue (ICB), characterized by its blue color in solution and a relatively shorter polymethine chain. A comparison of the ICG-ICB pair revealed important findings, including enhanced brightness of fluorescent dyes in heavy water that have emission bands exceeding 650 nm, and strong attenuation of the fluorescence enhancement when the dye is dehydrated upon binding to albumin protein. Chapters 4 and 5 detail molecular design strategies that produce a sterically "shielded" heptamethine dye family with near-infrared fluorescence. These dyes incorporate short ethylene glycol chains as "arms" to protect the polymethine core, thereby improving solubility, stability, aggregation behavior, pharmacokinetics, and biodistribution profiles. The versatile utility of this dye family was demonstrated by conducting a variety of assays that measured protein binding, metabolic activity, photophysical properties, and biodistribution in mouse cancer models. Chapter 6 delves into the realm of "multivalent" affinity-based probes aimed at enhancing probe design strategy. Here, a far-red Cy5 dye was conjugated with multiple benzenesulfonamide groups to target Carbonic Anhydrase IX (CA-IX) receptors, which are often overexpressed in hypoxic tumors. Imaging conducted on both 2D cell cultures and 3D tumor spheroids demonstrates stronger binding and brighter fluorescence for the multivalent probe compared to its monovalent counterpart.