Abstract

Fluorescence imaging in the second near-infrared window (NIR-II) holds promise for real-time deep tissue imaging. In this work, we investigated the NIR-II fluorescence properties of a liposomal formulation of indocyanine green (ICG), a FDA-approved dye that was recently shown to exhibit NIR-II fluorescence. Fluorescence spectra of liposomal-ICG were collected in phosphate-buffered saline (PBS) and plasma. Imaging studies in an Intralipid® phantom were performed to determine penetration depth. In vivo imaging studies were performed to test real-time visualization of vascular structures in the hind limb and intracranial regions. Free ICG, NIR-I imaging, and cross-sectional imaging modalities (MRI and CT) were used as comparators. Fluorescence spectra demonstrated the strong NIR-II fluorescence of liposomal-ICG, similar to free ICG in plasma. In vitro studies demonstrated superior performance of liposomal-ICG over free ICG for NIR-II imaging of deep (≥4 mm) vascular mimicking structures. In vivo, NIR-II fluorescence imaging using liposomal-ICG resulted in significantly (p < 0.05) higher contrast-to-noise ratio compared to free ICG for extended periods of time, allowing visualization of hind limb and intracranial vasculature for up to 4 hours post-injection. In vivo comparisons demonstrated higher vessel conspicuity with liposomal-ICG-enhanced NIR-II imaging compared to NIR-I imaging.

Highlights

  • Imaging studies in an Intralipid® phantom were performed to determine penetration depth

  • The NIR-I fluorescence intensity of liposomal-indocyanine green (ICG) measured over 80 days showed less than 10% drop in signal intensity demonstrating long-term stability of the formulation

  • Real-time near infrared imaging has applications in lymphangiography, and intraoperative imaging including the detection of occult vascular structures, sentinel lymph nodes and tumor margin detection[22,23,24,25,26,27,28,29]

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Summary

Introduction

Imaging studies in an Intralipid® phantom were performed to determine penetration depth. In vivo imaging studies were performed to test real-time visualization of vascular structures in the hind limb and intracranial regions. In vitro studies demonstrated superior performance of liposomal-ICG over free ICG for NIR-II imaging of deep (≥4 mm) vascular mimicking structures. NIR-II fluorescence imaging using liposomal-ICG resulted in significantly (p < 0.05) higher contrast-tonoise ratio compared to free ICG for extended periods of time, allowing visualization of hind limb and intracranial vasculature for up to 4 hours post-injection. In vivo comparisons demonstrated higher vessel conspicuity with liposomal-ICG-enhanced NIR-II imaging compared to NIR-I imaging. In vivo NIR-II imaging studies using ICG enabled the visualization of deep structures with higher contrast-to-noise ratio (CNR) than in the NIR-I window. In vivo studies were performed in nude mice to study the performance of liposomal-ICG as a NIR-II imaging agent for visualization of deep structures. Comparisons were made to cross-sectional imaging techniques (CT in the hind-limb and MRI in the brain)

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