700-nm Zwitterionic Near-Infrared Fluorophores for Dual-Channel Image-Guided Surgery

Abstract

Purpose: The purpose of this study was to develop a family of 700-nm zwitterionic pentamethine indocyanine near-infrared fluorophores that would permit dual-channel image-guided surgery.

Procedures: Three complementary synthetic schemes were used to produce novel zwitterionic chemical structures. Physicochemical, optical, biodistribution, and clearance properties were compared to Cy5.5, a conventional pentamethine indocyanine now used for biomedical imaging.

Results: ZW700-1a, ZW700-1b, and ZW700-1c were synthesized, purified, and analyzed extensively in vitro and in vivo. All molecules had extinction coefficients ≥199,000 M(-1) cm(-1), emission ≥660 nm, and stability ≥99 % after 24 h in warm serum. In mice, rats, and pigs, ≥80 % of the injected dose was completely eliminated from the body via renal clearance within 4 h. Either alone or conjugated to a tumor targeting ligand, ZW700-1a permitted dual-channel, high SBR, and simultaneous imaging with 800-nm NIR fluorophores using the FLARE® imaging system.

Conclusions: Novel 700-nm zwitterionic NIR fluorophores enable dual-NIR image-guided surgery.

Keywords: Image-guided surgery; Near-infrared fluorescence; Optical imaging; Signal-to-background ratio.

Figure 1. Synthetic schemes and 3D structures of ZW700-1 NIR fluorophores

a) Synthetic scheme for the three molecules studied. b) 3D structures and net charges of ZW700-1 and Cy5.5. Red: negative charge; blue: positive charge; gray: hydrophobic.

Figure 2. Live cell imaging and cytotoxicity of NIR fluorophores in C2C12 mouse myoblast cells

a) Shown are phase contrast and NIR images of each cell line tested at a concentration of 2 µM (exposure time = 200 msec). Scale bars = 100 µm. b) Cell viability was plotted 1 day post-treatment of each NIR fluorophore at a concentration of 2 µM (left) or 10 µM (right). Data are representative of N = 6 independent experiments per condition (mean ± S.D.). *P < 0.05.

Figure 3. In vivo clearance and elimination of ZW700-1 fluorophores in mice

a) 10 nmol of ZW700-1 NIR fluorophores were injected intravenously into 25 g CD-1 mice 4 h prior to imaging. Exposure time = 200 msec. Abbreviations used are: Bl, bladder; Du, duodenum; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Pa, pancreas; Sp, spleen. Scale bars = 1 cm. b) UPLC chromatography and ESI-TOF mass spectrometry of ZW700-1 fluorophores in mouse urine 4 h post-injection. Desalting microcolumns, packed with Poros R2 resin (Applied Biosystems, Framingham, MA), were used to remove excess salts and contaminants from urine samples. Data are representative of N = 3 independent experiments per condition.

Figure 4. In vivo clearance and elimination of ZW700-1 fluorophores in pigs

5 µmol of ZW700-1 NIR fluorophores were injected intravenously into 35 kg Yorkshire pigs 4 h prior to imaging. Exposure time = 200 msec. Abbreviations used are: Bl, bladder; Ki, kidneys; Li, liver; Sp, spleen, St, stomach, Ur, ureter. Scale bars = 1 cm. For blood clearance (%ID/g), blood half-life (mean ± 95% confidence intervals), and urine elimination of ZW700-1a and ICG in pig. Data are representative of N = 3 independent experiments per condition (mean ± S.D.). ***P < 0.001.

Figure 5. Simultaneous in vivo dual-channel NIR fluorescence imaging in the same animal

a) Renal vs. hepatobiliary clearance: 50 nmol of ZW700-1a and ICG were intravenously injected into a 250 g Sprague-Dawley rat 1 h prior to imaging (N =3). Kidneys, bladder, and ureters (arrows) were imaged using the 700 nm NIR channel (exposure time = 200 msec), while liver, duodenum, and bile duct (arrowhead) were imaged using the 800 nm (exposure time = 100 msec). Abbreviations used are: Bl, bladder; Du, duodenum; In, intestine; Ki, kidneys; Li, liver. b) Vasculature and tumor imaging: 10 nmol of BSA-ZW700-1a and 25 nmol of cRGD-ZW800-1 were injected into a 25 g xenograft tumor mouse 1 h and 4 h prior to imaging, respectively (N =3). Vasculature was imaged using the 700 nm NIR channel (exposure time = 200 msec), while integrin-αvβ3 overexpressing tumor (Tu; arrowheads) was imaged using the 800 nm channel (exposure time = 100 msec). The 700 nm and 800 nm images were pseudo-colored in red and green, respectively to generate the merged image. Scale bars = 1 cm.