Synthesis and Characterization of ICG-based Near-infrared Photoacoustic Contrast Agents

ABSTRACT Near-infrared photoacoustic imaging (NIR-PAI) integrates optical excitation with ultrasound detection to enable high-resolution, deep-tissue imaging by taking advantage of reduced light scattering and absorption in this spectral window. Despite its potential, clinical translation of contrast-enhanced NIR-PAI is limited by the scarcity of effective contrast agents. Indocyanine green (ICG), an FDA-approved NIR dye, is a strong candidate due to its biocompatibility and photoacoustic efficiency. However, its concentration-dependent aggregation, lack of facile targeting strategies, instability in aqueous environments, and low photostability result in variable signal, high background noise, and reduced reliability in vivo. To address these challenges, we developed three biocompatible ICG-based nanoprobe platforms amenable to facile, scalable synthesis: 5-arm DNA-ICG nanostructures (5-arm DNA-ICG), lipid-shelled ICG nanobubbles (ICG-NBs), and Azide-modified ICG J-aggregates (JAAZ). These platforms are designed to preserve ICG monomers or control aggregation, enabling enhanced NIR-PAI performance. Spectroscopic and photoacoustic analyses revealed consistent absorbance and photoacoustic profiles , showing enhanced signals compared to free ICG. The greatest improvement was observed for JAAZ, followed by ICG-NBs and 5-arm DNA-ICG. Photostability studies showed that JAAZ aggregation protects ICG from light-induced photodegradation, whereas monomer preservation in 5-arm DNA-ICG and ICG-NBs provides less protection and moderate signal stability. All three probes demonstrated stable performance under physiological conditions, achieved strong signal-to-noise ratios at depth and under tissue-mimicking conditions, and required markedly reduced probe concentrations to generate robust signals. Their modular architectures allow incorporation of targeting ligands, offering molecular specificity and multimodal functionality. Collectively, these contrast agent platforms provide noninvasive, deep-tissue molecular imaging and biosensing, with strong potential for future preclinical and clinical translation, and represent a promising alternative to free ICG for biomedical applications.