Quantitative Imaging of Lymphatic Function with Liposomal Indocyanine Green

Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich, Zurich, Switzerland.
Cancer Research (Impact Factor: 9.33). 09/2010; 70(18):7053-62. DOI: 10.1158/0008-5472.CAN-10-0271
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Lymphatic vessels play a major role in cancer progression and in postsurgical lymphedema, and several new therapeutic approaches targeting lymphatics are currently being developed. Thus, there is a critical need for quantitative imaging methods to measure lymphatic flow. Indocyanine green (ICG) has been used for optical imaging of the lymphatic system, but it is unstable in solution and may rapidly enter venous capillaries after local injection. We developed a novel liposomal formulation of ICG (LP-ICG), resulting in vastly improved stability in solution and an increased fluorescence signal with a shift toward longer wavelength absorption and emission. When injected intradermally to mice, LP-ICG was specifically taken up by lymphatic vessels and allowed improved visualization of deep lymph nodes. In a genetic mouse model of lymphatic dysfunction, injection of LP-ICG showed no enhancement of draining lymph nodes and slower clearance from the injection site. In mice bearing B16 luciferase-expressing melanomas expressing vascular endothelial growth factor-C (VEGF-C), sequential near-IR imaging of intradermally injected LP-ICG enabled quantification of lymphatic flow. Increased flow through draining lymph nodes was observed in mice bearing VEGF-C-expressing tumors without metastases, whereas a decreased flow pattern was seen in mice with a higher lymph node tumor burden. This new method will likely facilitate quantitative studies of lymphatic function in preclinical investigations and may also have potential for imaging of lymphedema or improved sentinel lymph detection in cancer.

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Available from: Paola Luciani, May 07, 2015
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    • "Liposomes were prepared by a film hydration/extrusion method [25]. In brief, egg PC and cholesterol were dissolved (60/40% molar ratio, total lipid concentration = 2.5 mM) with por (1.5 mg/mL final concentration) in chloroform and dried overnight. "
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    • "Additional controversy also remains regarding the most appropriate fluorophore for NIR lymphatic imaging. Some studies are beginning to explore novel probes with higher quantum yields that specifically target lymphatic vessels (Proulx et al., 2010, 2013; Davies-Venn et al., 2011), but the most commonly used probe to date has been indocyanine green (ICG). Despite the low quantum yield of the molecule, ICG remains the hallmark of NIR lymphatic imaging because it is FDA approved for use in humans and represents the most likely probe for the development of a point-of-care diagnostic. "
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    ABSTRACT: Introduction: Near-infrared (NIR) imaging has emerged as a novel imaging modality for assessing lymphatic function in vivo. While the technique has provided quantitative data previously unavailable, questions remain in regards to the spatiotemporal capabilities of the approach. We address three of the more important issues here using the rodent tail, one of the most widely utilized in vivo model systems in the lymphatic literature. Specifically we demonstrate (1) the transient vs. steady state response of lymphatics to tracer injection, (2) the functional characteristics of multiple collecting vessels draining the same tissue space in parallel, and (3) the long-term consequences of fluorescent tracers on lymphatic function to repeated functional measurements. Methods: Rat tails were imaged with NIR and metrics of function were calculated for both collecting vessels that drain the tail. A nitric oxide donor cream (GTNO) was applied to the tail. Additionally, two different NIR dyes, indocyanine green (ICG) and LI-COR IRDye 800CW PEG, were utilized for function imaging at the time of initial injection and at 1, 2, and 4 week follow-up time points after which both draining lymph nodes were harvested. Results and Discussion: Significant differences were found between the two collecting vessels such that the vessel first showing fluorescence (dominant) produced enhanced functional metrics compared to the second vessel (non-dominant). GTNO significantly reduced lymphatic function in the non-dominant vessel compared to the dominant. ICG remained visible in the tail for 2 weeks after injection and was accompanied by significant losses in lymphatic function and enlarged draining lymph nodes. The Licor tracer also remained visible for 2 weeks. However, the dye produced significantly lower effects on lymphatic function than ICG, and lymph nodes were not enlarged at any time point, suggesting that this may be a more appropriate contrast agent for longitudinal lymphatic imaging.
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