Research ArticlesDegradation Kinetics of Indocyanine Green in Aqueous Solution
Introduction
Current studies of the near‐infrared (IR) fluorescence agents have proven their usefulness in numerous analytical and diagnostic applications. These agents strongly absorb in the near‐IR region of the spectrum leading to a strong near‐IR fluorescence emission, thus enabling their precise and accurate detection. Their use as biomarkers for in vivo imaging exhibits a particular advantage of enhanced selectivity. Because most of the biomolecules neither absorb nor emit in the near‐IR region, the signals emitted by the near‐IR fluorescence agents are relatively free of intrinsic background interference.
Indocyanine green (ICG) is a tricarbocyanine dye, which absorbs and emits in the near‐IR region of spectrum. It has been approved by the United States Food and Drug Administration for medical diagnostic studies, and has been widely used for the evaluation of cardiac output, liver function, microcirculation of skin flaps, visualization of retinal and choroidal vasculatures, pharmacokinetic analysis, object localization in tissue, tissue welding, fluorescence probing of enzyme and proteins. All the above‐mentioned uses of ICG are based on its fluorescing ability.1,2 The chemical structure of ICG is shown in Figure 1.
Moreover, ICG has a great potential for application in photodynamic therapy, which also utilizes its fluorescing ability.3 An important motivation for using ICG in such studies is its strongest absorption band around 800 nm and its most intense emission around 820 nm.4,5 These are the wavelengths for which the blood and other tissues are relatively transparent and the penetration depth of light in biological tissue is the highest.6 As a result, ICG provides enhanced specificity for photodynamic tumor therapy.
For all the above‐mentioned applications, ICG has to be administered in the form of aqueous solutions. There are many reports about the instability of ICG in aqueous solutions. In aqueous solutions, ICG undergoes physicochemical transformations such as aggregation and irreversible degradation.1,7 Such changes result in discoloration, decreased light absorption, decreased fluorescence, and a shift in the wavelength of maximum absorption. It was reported that pH has an important role in ICG degradation in water.8
However, there is still a lack of knowledge about the degradation kinetics of ICG, the effect of factors such as concentration, light exposure, and temperature on its degradation kinetics. This type of information is very critical for the advancement of the application of ICG in biomedical and pharmaceutical sciences. Moreover, earlier studies on ICG degradation used absorption spectroscopic measurements, which comprised complex absorption and transmission spectrum analysis and lacked selectivity. Because the practical use of ICG lies in its fluorescence ability, the stability studies should be based on the fluorescence measurements, which are relatively more accurate and selective.
Therefore, the objective of our present study is to extend the insight into the degradation behavior of ICG in aqueous solutions. The present study used a near‐IR fluorescence technique to monitor ICG concentrations in aqueous solutions. The effect of ICG concentration on its fluorescence spectrum was studied. The order and rate of degradation of ICG were analyzed, and the influences of light exposure, type of light, temperature, and ICG concentration on degradation were also studied. We believe this study may be useful as a reference in the research of the stability of other cyanine dyes that undergo degradation in aqueous solutions.
Section snippets
Materials
ICG (IR‐25, laser grade) was obtained from Fisher Scientific (Pittsburgh, PA) and ICG sodium iodide salt (Diagnogreen® Injection) was obtained from Daiichi Pharmaceutical (Tokyo, Japan). Screw capped transparent centrifuge tubes were obtained from VWR International (West Chester, PA). Distilled water (by reverse osmosis), pH 7, filtered by 0.22‐μ syringe filter (Syrfil; MF Whatman Inc., Clifton, NJ) was used for all the studies.
Fluorescence Spectroscopy Instrumentation
To obtain the kinetic degradation data, a K‐2 multifrequency
Influence of ICG Concentration on Its Fluorescence Spectra and Intensity
Figure 2 shows the plot of peak fluorescence intensities versus ICG concentration. The plot reflects the increase in peak fluorescence intensity of ICG with increase in ICG concentration up to a maximum value of 2 μg/mL after which a further increase in ICG concentration causes a gradual decrease in the peak fluorescence intensity. This phenomenon is due to the formation of weakly fluorescent ICG molecular aggregates at high concentrations, concentration quenching (i.e., self‐quenching), and
Conclusion
This investigation shows that the degradation of ICG in aqueous solution follows (pseudo) first‐order kinetics. At concentrations <2 μg/mL, the emission spectrum of ICG shows an increase in peak fluorescence intensity with a rise in concentration. At concentrations >2 μg/mL, it is observed that an increase in concentration caused a decrease in peak fluorescence intensity with a right shift in peak wavelength due to the quenching effects. The degradation of ICG in aqueous solutions is
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