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. Jun-Jul 2018;26(5-6):494-504.
doi: 10.1080/1061186X.2018.1433681. Epub 2018 Feb 12.

Indocyanine Green Nanoparticles Undergo Selective Lymphatic Uptake, Distribution and Retention and Enable Detailed Mapping of Lymph Vessels, Nodes and Abnormalities

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Indocyanine Green Nanoparticles Undergo Selective Lymphatic Uptake, Distribution and Retention and Enable Detailed Mapping of Lymph Vessels, Nodes and Abnormalities

John C Kraft et al. J Drug Target. .
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Abstract

The distributed network of lymph vessels and nodes in the body, with its complex architecture and physiology, presents a major challenge for whole-body lymphatic-targeted drug delivery. To gather physiological and pathological information of the lymphatics, near-infrared (NIR) fluorescence imaging of NIR fluorophores is used in clinical practice due to its tissue-penetrating optical radiation (700-900 nm) that safely provides real-time high-resolution in vivo images. However, indocyanine green (ICG), a common clinical NIR fluorophore, is unstable in aqueous environments and under light exposure, and its poor lymphatic distribution and retention limits its use as a NIR lymphatic tracer. To address this, we investigated in mice the distribution pathways of a novel nanoparticle formulation that stabilises ICG and is optimised for lymphatic drug delivery. From the subcutaneous space, ICG particles provided selective lymphatic uptake, lymph vessel and node retention, and extensive first-pass lymphatic distribution of ICG, enabling 0.2 mm and 5-10 cell resolution of lymph vessels, and high signal-to-background ratios for lymphatic vessel and node networks. Soluble (free) ICG readily dissipated from lymph vessels local to the injection site and absorbed into the blood. These unique characteristics of ICG particles could enable mechanistic studies of the lymphatics and diagnosis of lymphatic abnormalities.

Keywords: Lymphatic drug delivery; indocyanine green; lymphatic fluorescence imaging; nanoparticles; near-infrared fluorescence imaging; small molecules.

Conflict of interest statement

Disclosure statement

The authors report no declarations of interest.

Figures

Figure 1
Figure 1
Comparison of free ICG, ICG particle and quantum dot (QD) clearance from the SC foot injection site. (A) Representative NIR fluorescence images of the injection site (N = 3–5). (B) Time course of the foot SC injection site NIR fluorescence intensity (arbitrary units, a.u.). Rate constants (min−1) and R2 values for trendlines are listed. (C) Schematic representation of the superficial lymphatic vessels and nodes (popliteal, sciatic, subiliac and axillary) on the right side of the mouse in relation to the dorsal right hind foot SC injection site.
Figure 2
Figure 2
Comparison of ICG localisation following SC dosing of free ICG or ICG particles. NIR fluorescence images (top three rows) and corresponding bright field images with false colour fluorescence overlay (bottom row) of the ventral right hind leg muscle tissue and saphenous vein (arrows) with skin removed 1, 5, 30 and 60 min post-SC foot injection of (A–C, G) free ICG or (D–F, H, I) ICG particles.
Figure 3
Figure 3
Time course of ICG distribution in the lymphatic system following a single SC dose of free ICG or ICG particles. NIR fluorescence images (top) and corresponding bright field images with false colour fluorescence overlay (bottom) showing the lymphatic distribution of ICG at 1, 5 and 30 min following SC dosing of (A) free ICG or (B) ICG particles. Comparison of the ICG fluorescence intensity versus distance from the SC injection site of the lymph node or thoracic lymph duct at (C) 1 min, (D) 5 min and (E) 30 min following SC injection of free ICG (open circles) or ICG particles (solid circles). The structures in the lymphatics that were assayed and their approximate distance away from the right hind foot SC injection site, which is listed on the x-axis of (C–E), are as follows: popliteal LN (2.0 cm), sciatic LN (2.5 cm), medial iliac LN (3.8 cm), lumbar LN (5.4 cm), inferior thoracic lymph duct (5.7 cm), medial thoracic lymph duct (6.5 cm) and superior thoracic lymph duct (7.3 cm). *, p values <.05.
Figure 4
Figure 4
(A) ICG particles detected a small lymph vessel adjacent to blood vessels. NIR fluorescence image of mouse skin opened with ICG particles post-SC foot injection tracing a small lymph vessel (stemmed arrows) connecting the right subiliac and axillary LNs without egressing from the lymph vessel into nearby tissue or the parallel dark blood vessel (non-stemmed arrows). Scale bar, 3 mm. (B) Lymphatic retention of ICG particles permitted detection of a lymphatic abnormality. NIR fluorescence image of ICG particles in the hind limbs of a mouse exhibiting abnormal, enlarged afferent popliteal lymph vessels (*). H&E histology (top) and anti-podoplanin immunohistochemistry (bottom) of control (left) and abnormal (right) lymph vessels that correspond to the locations in the mouse indicated by arrows. Histological analysis shows perivascular and intravascular mononuclear cell accumulations for the abnormal lymph vessels (right) but not for the normal lymph vessels (left).

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