Sterically Shielded Hydrophilic Analogs of Indocyanine Green

Abstract

A modular synthetic process enables two or four shielding arms to be appended strategically over the fluorochromes of near-infrared cyanine heptamethine dyes to create hydrophilic analogs of clinically approved indocyanine green. A key synthetic step is the facile substitution of a heptamethine 4'-Cl atom by a phenol bearing two triethylene glycol chains. The lead compound is a heptamethine dye with four shielding arms, and a series of comparative spectroscopy studies showed that the shielding arms (a) increased dye photostability and chemical stability and (b) inhibited dye self-aggregation and association with albumin protein. In mice, the dye cleared from the blood primarily through the renal pathway rather than the biliary pathway for ICG. This change in biodistribution reflects the much smaller hydrodynamic diameter of the shielded hydrophilic ICG analog compared to the 67 kDa size of the ICG/albumin complex. An attractive feature of versatile synthetic chemistry is the capability to systematically alter the dye's hydrodynamic diameter. The sterically shielded hydrophilic ICG dye platform is well-suited for immediate incorporation into dynamic contrast-enhanced (DCE) spectroscopy or imaging protocols using the same cameras and detectors that have been optimized for ICG.

Figure 1.

(a) Absorption spectra (3 μM in PBS, pH 7.4). (b) Photobleaching of ICG and its analogs (2 μM in pH 7.4 PBS irradiated by a 0.5 mW/cm² Xenon lamp with a 620 nm long-pass filter).

Figure 2.

Energy-minimized molecular model of shielded dye 3, red bonds for shielding arms, blue bonds for heptamethine fluorochrome; H: white atoms; O: red atoms; N: deep blue atoms; S: yellow atoms.

Figure 3.

(a) Representative overlaid brightfield and fluorescence ex vivo images of major organs of BALB/c mice sacrificed 2 h after retro-orbital injection of either ICG (top) or 3 (bottom). Scale is in arbitrary fluorescence units. (b) Biodistribution of ICG and 3 in BALB/c mice. Note that this data does not include the large fraction of probe 3 excreted as urine. *p < 0.1, **p < 0.05, otherwise no statistically significant difference.

Scheme 1.

Chemical Structures of ICG, IR820, IRDye800CW, and CW800-SO₃

Scheme 2.

Chemical Structures and Cartoon Representations of ICG Analogs in This Study

Scheme 3.

Synthesis of ICG Analogs

Scheme 4.

Substitution of the 4’-phenoxy group of CW800-SO₃ by GSH