Structure-inherent targeting of near-infrared fluorophores for parathyroid and thyroid gland imaging

Abstract

The typical method for creating targeted contrast agents requires covalent conjugation of separate targeting and fluorophore domains. In this study, we demonstrate that it is possible to create near-infrared (NIR) fluorophores with different tissue specificities driven by their inherent chemical structures. Thus, a single compact molecule performs both targeting and imaging. We use this strategy to solve a major problem in head and neck surgery: the identification and preservation of parathyroid and thyroid glands. We synthesized 700-nm and 800-nm halogenated fluorophores that show high uptake into these glands after a single intravenous (IV) injection of 0.06 mg kg(-1) in a pig. By using a dual-channel NIR imaging system, we observed-in real time and with high sensitivity-the unambiguous distinction of parathyroid and thyroid glands simultaneously in the context of blood and surrounding soft tissue. This novel technology lays a foundation for performing head and neck surgery with increased precision and efficiency along with potentially lower morbidity, and it provides a general strategy for developing targeted NIR fluorophores.

Figure 1

(a) Synthetic schemes for T700 and T800 NIR fluorophores, and (b) their physicochemical and optical properties in 100% serum, pH 7.4. In silico calculations of logD at pH 7.4 and total polar surface area were calculated using Marvin and JChem calculator plugins (ChemAxon, Budapest, Hungary).

Figure 2

In vivo thyroid targeting of (a) T700 and (b) T800 NIR fluorophores in mice. Each fluorophore was intravenously injected into 20 g CD-1 mice (10 nmol; 0.2 mg kg⁻¹) 1 h and 4 h prior to imaging. SBR was calculated by the fluorescence intensity of thyroid tissues versus the signal intensity of neighboring muscle obtained over the imaging period. All NIR fluorescence images have identical exposure and normalizations. Abbreviations used are: SG, salivary glands and TG, thyroid glands (arrows). Scale bars = 1 cm. Images are representative of n = 3 independent experiments at 1 h post-injection.

Figure 3

In vivo parathyroid and thyroid imaging using T700-F and T800-F in pigs. 5 μmol (0.06 mg kg⁻¹) of T700-F and T800-F were injected intravenously into 35 kg Yorkshire pigs 5 h prior to imaging. All NIR fluorescence images have identical exposure and normalizations. SBR was calculated by the fluorescence intensity of each tissue versus the signal intensity of neighboring muscle obtained over the period of imaging time. Parathyroid signal to thyroid signal ratio (PTR) curves were determined from individual SBR curves. Abbreviations used are: La, larynx; PG, parathyroid glands (arrowheads); TG, thyroid gland (arrows), and Th, thymus. Scale bars = 1 cm. Each data point is the mean ± s.d. from n = 3 animals.

Figure 4

(a) Dual-channel in vivo fluorescence imaging using T700-F and T800-F in the same rat. 0.2 μmol (0.35 mg kg⁻¹) of T800-F was intravenously injected into a 250 g SD rat 24 h prior to imaging, followed by 0.2 μmol of T700-F injected 6 h prior to imaging. Abbreviations used are: Es, esophagus; PG, parathyroid glands (arrowheads); SG, salivary glands; TG, thyroid glands (arrows); and Tr, trachea. Scale bars = 1 cm. (b) H&E and NIR imaging of resected parathyroid and thyroid tissues from (a). Scale bars = 300 μm. All NIR fluorescence images for each condition have identical exposure times and normalizations. Pseudo-colored red and green colors were used for 700 nm and 800 nm channel images, respectively, in the color-NIR merged image.

Figure 5

(a) Dual-channel in vivo fluorescence imaging using T700-F and T800-F in the same pig. 5 μmol (0.06 mg kg⁻¹) of T800-F was intravenously injected into a 35 kg Yorkshire pig 4 h prior to imaging, followed by 5 μmol of T700-F injected 2 h later. Abbreviations used are: La, larynx; PG, parathyroid glands (arrowheads); and TG, thyroid gland (arrows). Scale bars = 1 cm. (b) H&E and NIR imaging of resected parathyroid and thyroid tissues from (a). Scale bars = 300 μm. All NIR fluorescence images have identical exposure and normalizations. Pseudo-colored red and green colors were used for 700 nm and 800 nm channel images, respectively, in the color-NIR merged image.