Deuterated Indocyanine Green (ICG) with Extended Aqueous Storage Shelf-Life: Chemical and Clinical Implications

Abstract

Indocyanine Green (ICG) is a clinically approved near-infrared fluorescent dye that is used extensively for various imaging and diagnostic procedures. One drawback with ICG is its instability in water, which means that reconstituted clinical doses have to be used very shortly after preparation. Two deuterated versions of ICG were prepared with deuterium atoms on the heptamethine chain, and the spectral, physiochemical, and photostability properties were quantified. A notable mechanistic finding is that self-aggregation of ICG in water strongly favors dye degradation by a photochemical oxidative dimerization reaction that gives a nonfluorescent product. Storage stability studies showed that replacement of C-H with C-D decreased the dimerization rate constant by a factor of 3.1, and it is likely that many medical and preclinical procedures will benefit from the longer shelf-lives of these two deuterated ICG dyes. The discovery that ICG self-aggregation promotes photoinduced electron transfer can be exploited as a new paradigm for next-generation photodynamic therapies.

Keywords: cyanines; deuterium; dyes/pigments; fluorescent probes; imaging agents.

Scheme 1

Chemical structures of ICG, [D₅]‐ICG, and [D₇]‐ICG.

Scheme 2

Three distinct degradation pathways for aqueous ICG in the presence of air and light. Pathway a: double‐bond cleavage to produce carbonyl‐containing fragments. Pathway b: truncation to produce pentamethine homologue. Pathway c: oxidative dimerization to produce 1.

Scheme 3

Syntheses of [D₅]‐ICG and [D₇]‐ICG.

Figure 1

a) Partial ¹H NMR spectra (500 MHz, [D₄]methanol, 25 °C) showing differences in stability for separate stock solutions of ICG or [D₅]‐ICG in water (1.0 mM) at room temperature. The spectra show a decrease in the peak for −CH₂SO₃ ⁻ protons at δ=2.91 ppm for ICG or [D₅]‐ICG and an increase in the corresponding peak at δ=2.80 ppm for oxidative dimer 1 or [D₈]‐1. b) Speciation plots showing the change in weight percentage with storage time. Each plot was fit to a pseudo‐second‐order dimerization reaction model. c) Curve fitting of the data to give the ratio of rate constants (k _H/k _D) as a deuterium kinetic isotope effect in water (1.0 mM) at room temperature (22 °C).

Scheme 4

Different major pathways for reaction of: a) ICG monomer with photosensitized singlet oxygen in any solvent, or b) excited state ICG in H‐aggregate with molecular oxygen. c) Proposed mechanism for photosensitized oxidative dimerization of ICG to form 1.[ ²⁷ , ⁵⁴ ]

Figure 2

NIR fluorescence images (λ _ex: 745 nm, λ _em: 850 nm) of two mouse phantoms with heart portals containing FBS and an aliquot from a 1.0 mM aqueous stock solution of either ICG or [D₅]‐ICG (final dye concentration, 2 μM). The top pair of phantoms contain freshly prepared stock solutions and the bottom pair contain 1.0 mM aqueous stock solutions that had been stored for 3 days at room temperature (22 °C). The fluorescence intensity scale is in arbitrarily units. The graphs compare normalized mean pixel intensity (MPI) for the fluorescent heart regions within each pair of mouse phantom images. The graph error bars are too small to visualize.