Revisiting Indocyanine Green: Effects of Serum and Physiological Temperature on Absorption and Fluorescence Characteristics

Abstract

Abstract-Indocyanine green (ICG) remains as the only near infrared dye approved by the FDA. Despite its long history of usage in clinical medicine, a systematic study of the effects of serum proteins at physiologically relevant levels and temperature on absorption and fluorescence characteristics of ICG has been missing. We incubated ICG at concentrations in the range of 0.6-25.8 μM in McCoy's 5a cell culture medium, without and with supplemental fetal bovine serum (FBS) at 5% and 10% levels. Our analyses of absorption and fluorescence spectra indicate that the peak absorbance of ICG associated with its monomeric form increases in the presence of FBS. For example, at ICG concentration of 25.8 μM, the monomer absorbance is increased by nearly 100% in the presence of 10% FBS. Similarly, there is an increase in the relative fluorescence quantum yield of ICG, by as much as nearly 3.5 times in the presence of FBS. When incubated at 37• C, the presence of FBS in the cell culture medium helps maintain the monomeric absorption of ICG and sustain the increased fluorescence emission. We offer explanations to describe the possible photophysical mechanisms underlying the observed effects and discuss the importance of these results to in-vivo applications of ICG.

Near-Infrared-Fluorescent Erythrocyte-Mimicking Particles: Physical and Optical Characteristics

“…The spectral features of ICG provide insight into the conformational states of ICG present within NETs. ICG can be in either a monomeric form, indicated by an absorption peak at 805 nm, or in an H-like aggregated form [73,74], indicated by a secondary absorption peak at 740 nm. We define the parameter  as the ratio of the monomeric ICG absorbance to that of the H-like aggregate form of ICG.…”

“…The absorption shoulder in the spectral range of 720-760 nm corresponds to the H-like aggregate form of ICG [54,74].…”

“…This sandwich-like, - stacking of individual ICG molecules results in interaction of the individual transition dipole moments that raises the excited-state energy to produce a blueshifted absorption peak [49,73,74]. In water, even relatively low concentrations of ICG (> 10 M) will begin to form Haggregates [74,85].…”

Photoacoustic ratiometric assessment of mitoxantrone release from theranostic ICG-conjugated mesoporous silica nanoparticles

“…Among the NIR probes, ICG is one of the most employed for PAI applications because it is already FDA approved for human diagnosis and it has a good safety profile. 7 However, its in vivo use is hampered by two important issues, i.e. i) ICG is strongly sequestered by Human Serum Albumin (HSA), 8 with a consequent removal from the nanoprobe and ii) the photobleaching of ICG due to interaction with microenvironmental species (e.g.…”

“…O2 and water of tissues). 7 The encapsulation of ICG inside nanocarriers (liposomes, micelles, lipidots, SWCN, etc…) has been exploited to enhance photostability of the molecule and prevent HSA binding, [9][10][11][12][13][14] although a competition study with HSA is often omitted. Notably, we have recently shown that the encapsulation of ICG into MSNs pores increases the photoacoustic effect of ca.…”