Novel ICG-based Nanoprobes for Near-infrared Photoacoustic Imaging

Introduction:: Photoacoustic imaging (PA) is a non-invasive technique that relies on the absorption of laser light and the subsequent thermal expansion of biological tissues for generating ultrasound signals. To reduce photon scattering in biological tissues and enhance imaging depth, near-infrared (NIR) illumination is employed in PA imaging. However, the adoption of this technology and clinical translation is hindered due to a lack of NIR-PA contrast agents.

Indocyanine green (ICG) dye is a viable candidate due to its NIR optical absorption, biocompatibility and approval by the FDA, and demonstrated use in PA imaging. However, ICG has a few shortcomings, including concentration-dependent aggregation and a lack of targeted labeling. Therefore, solutions that package ICG to facilitate stable optical characteristics and conjugation for targeting are critically needed.

This investigation focuses on three diverse approaches to address this need. We present the optical and photoacoustic characterization of three new ICG-based contrast platforms, namely 1D (1-arm) DNA-ICG nano-construct, 2D (5-arm) DNA-ICG nano-construct, and J-aggregates and compared them to free ICG. The DNA-based nanostructures were synthesized using the DNA origami technique (programmable self-assembly) to produce nano-scaffolds, which were templated with free ICG molecules yielding four dye molecules on each 1D construct and 15 dye molecules on each 2D nano-construct. J-aggregates are brick-like assemblies of ICG that provide a potential alternative to monomeric ICG. These platforms possess distinct optical and photoacoustic signatures and can be readily conjugated with targeting molecules, antibodies, or aptamers.


Materials and Methods:: To synthesize the DNA-based probes, ssDNA oligonucleotides modified with cholesterol or ICG moieties were assembled by annealing at a temperature range of 90°C to 4°C for over two hours. Azide-modified ICG J-aggregates (JAAZ) were synthesized by incubating solutions with varying molar ratios of ICG and ICG-azide dye at 60°C with various KCl concentrations. The optical absorption of the probe solutions was recorded using UV-Vis-NIR spectroscopy in phosphate-buffered saline (PBS) at concentrations of 15, 30, 60, 90, and 120 µM, and fluorescence spectra were measured for concentrations above for the peak fluorescence region of ~775 – 850 nm via a fluorescence plate reader and compared to plain ICG. The PA signal of various probe concentrations diluted in PBS was also measured at their peak excitation wavelength. To simulate intravenous delivery, the stability of probes in blood was characterized by mixing probe solutions in PBS with whole sheep's blood in a tube phantom and then measuring PA signals at their peak excitation wavelength at concentrations of 7.5, 15, 30, 45, and 60 µM, normalized to the PA of the mixture of PBS/blood. Additionally, the PA signals for the four contrast agents were recorded at 690 – 945 nm wavelengths.


Results, Conclusions, and Discussions:: The absorption spectra of ICG, DNA nano-constructs, and J-aggregate ICG platform were examined in PBS. The peak absorption of monomeric ICG remained constant at 780 nm but was slightly red-shifted (~805 nm) when incorporated into each of the DNA-NP platforms. JAAZ showed a strong, red-shifted absorption peak at 895 nm.

Fluorescence spectrometry measurements revealed an increase in fluorescence intensity as a function of ICG concentration in 1-arm and 5-Arm probes. However, JAAZ showed almost no fluorescence upon optical excitation, suggesting strong fluorescent quenching effects, which are conducive to PA-based sensing and imaging.

The highest PA signal amplitude was recorded from JAAZ, followed by the 5-Arm and 1-arm probes, then the free ICG. The most pronounced gains in J-aggregates PA signal amplitude occurred at relatively high concentrations ( >90 µM). This is in stark contrast to free ICG, which does not have appreciable PA signal amplitude gains at concentrations higher than 60 µM due to the dimerization effects of ICG at those concentrations. The concentration-dependent PA signals from 1-arm and 5-arm probes excited at the monomeric excitation peak (805 nm) demonstrate that the DNA probe is inhibiting the dimerization of the ICG molecules.

Our results confirmed the photoacoustic spectra from these contrast agents agree with absorbance measurements. Concentration-dependence of the PA signal of all probes was conserved when nano-probes were introduced into the blood, similar to that observed when the probes were diluted in PBS. When probe concentration exceeded 30 µM, PA signal amplitude was greater than that obtained from whole blood. JAAZ showed a PA signal stronger than blood even at the lowest concentration and had the highest amplitude in the PA signal compared to plain ICG and DNA nanoprobes.

We examined the feasibility of three ICG-based nano-probes as potential candidates for NIR-PA imaging. They are size-tunable, compatible with common bio-functionalization techniques, and optically and structurally stable in physiological environments. They also exhibit stronger NIR absorption and PA signal intensity than ICG alone, enabling deeper tissue imaging. For instance, the PA signal from JAAZ at 60 µM was slightly greater than the PA signal from free ICG at 120 µM.

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