Organic Alternatives to Quantum Dots for Intraoperative Near-Infrared Fluorescent Sentinel Lymph Node Mapping

Abstract

Intraoperative near-infrared (NIR) fluorescence imaging provides the surgeon with real-time image guidance during cancer and other surgeries. We have previously reported the use of NIR fluorescent quantum dots (QDs) for sentinel lymph node (SLN) mapping. However, because of concerns over potential toxicity, organic alternatives to QDs will be required for initial clinical studies. We describe a family of 800 nm organic heptamethine indocyanine-based contrast agents for SLN mapping spanning a spectrum from 775 Da small molecules to 7 MDa nanocolloids. We provide a detailed characterization of the optical and physical properties of these contrast agents and discuss the advantages and disadvantages of each. We present robust methods for the covalent conjugation, purification, and characterization of proteins with tetra-sulfonated heptamethine indocyanines, including mass spectroscopic site mapping of highly substituted molecules. One contrast agent, NIR fluorescent human serum albumin (HSA800), emerged as the molecule with the best overall performance with respect to entry to lymphatics, flow to the SLN, retention in the SLN, fluorescence yield and reproducibility. This preclinical study, performed on large animals approaching the size of humans, should serve as a foundation for future clinical studies.

Sentinel Lymph Node Mapping of Invasive Urinary Bladder Cancer in Animal Models Using Invisible Light

“…Optical imaging using NIR fluorescent lymphatic tracers provides real-time assessment of lymphatic flow from the point of injection to the SLN(s), relatively high sensitivity due to the penetrating ability of NIR photons, and elimination of the need for radioactivity. NIRFI has been validated in large animal models for SLN mapping of skin [16,17], lung [21], esophagus [22], pleural space [22], GI tract [16,21], and spontaneous melanoma [23]. Our study of invasive bladder cancer confirms that the anatomy of lymphatic flow is highly variable among individuals, and can involve structures such as the urachus and presacral nodes, and that SLN mapping must be performed on an individual basis.…”

“…Canine bladder cancer closely resembles InvTCC in humans with regard to histopathological characteristics, cellular and molecular features, and biological behavior including sites and frequency of metastasis [15]. Of particular importance for translation of our results into human studies, we have recently described a formulation of two clinically available agents that can be used for SLN mapping in humans [16].…”

“…Human serum albumin conjugated with three molecules of IRDye ™ 800CW (HD, 7.4 nm; LI-COR; Lincoln, NE, USA) [16], HSA800, and NIR fluorescent quantum dots (NIRQDs) with an oligomeric phosphine coating (HD, 15-20 nm) [17] have been described previously. Peak excitation and emission wavelengths of HSA800 in serum were 784 and 802 nm, respectively, and per fluorophore quantum yield was 12%.…”

The use of fluorescent dyes and probes in surgical oncology

“…However, there is a considerable range in the quantum yields of the dyes. ICG and Irdye800 CW have the lowest QY (respectively 0.13 and 0.15) 7 , followed by Cy5.5 and Cy7 (respectively 0.25 and 0.27). The QDs have the highest QY (0.50-0.60), making them highly fluorescent.…”

“…29 For successful application of fluorescent dyes or probes, three features are important: median hydrodynamic diameter, absence of surface charge, and high contrast generation. 7 In the paragraphs below the different dyes and probes and their modes of functioning are discussed.…”