Dual-modality imaging with 99mTc and fluorescent indocyanine green using surface-modified silica nanoparticles for biopsy of the sentinel lymph node: an animal study

Abstract

Background: We propose a new approach to facilitate sentinel node biopsy examination by multimodality imaging in which radioactive and near-infrared (NIR) fluorescent nanoparticles depict deeply situated sentinel nodes and fluorescent nodes with anatomical resolution in the surgical field. For this purpose, we developed polyamidoamine (PAMAM)-coated silica nanoparticles loaded with technetium-99m (99mTc) and indocyanine green (ICG).

Methods: We conducted animal studies to test the feasibility and utility of this dual-modality imaging probe. The mean diameter of the PAMAM-coated silica nanoparticles was 30 to 50 nm, as evaluated from the images of transmission electron microscopy and scanning electron microscopy. The combined labeling with 99mTc and ICG was verified by thin-layer chromatography before each experiment. A volume of 0.1 ml of the nanoparticle solution (7.4 MBq, except for one rat that was injected with 3.7 MBq, and 1 μg of an ICG derivative [ICG-sulfo-OSu]) was injected submucosally into the tongue of six male Wistar rats.

Results: Scintigraphic images showed increased accumulation of 99mTc in the neck of four of the six rats. Nineteen lymph nodes were identified in the dissected neck of the six rats, and a contact radiographic study showed three nodes with a marked increase in uptake and three nodes with a weak uptake. NIR fluorescence imaging provided real-time clear fluorescent images of the lymph nodes in the neck with anatomical resolution. Six lymph nodes showed weak (+) to strong (+++) fluorescence, whereas other lymph nodes showed no fluorescence. Nodes showing increased radioactivity coincided with the fluorescent nodes. The radioactivity of 15 excised lymph nodes from the four rats was assayed using a gamma well counter. Comparisons of the levels of radioactivity revealed a large difference between the high-fluorescence-intensity group (four lymph nodes; mean, 0.109% ± 0.067%) and the low- or no-fluorescence-intensity group (11 lymph nodes; mean, 0.001% ± 0.000%, p < 0.05). Transmission electron microscopy revealed that small black granules were localized to and dispersed within the cytoplasm of macrophages in the lymph nodes.

Conclusion: Although further studies are needed to determine the appropriate dose of the dual-imaging nanoparticle probe for effective sensitivity and safety, the results of this animal study revealed a novel method for improved node detection by a dual-modality approach for sentinel lymph node biopsy.

Figure 1

PAMAM-coated silica nanoparticles loaded with ^99mTc and ICG.

Figure 2

SEM and TEM images of silica nanoparticles, distribution of particle sizes, and combined labeling. The mean diameter of the PAMAM-coated silica nanoparticles was 20 to 50 nm, as evaluated by scanning electron microscopy (A) and transmission electron microscopy (B). The distribution of the particle sizes was measured using a laser-scattering particle-size analyzer (C). The peak of the distribution was 30 nm in ethanol solution. Combined labeling with both ^99mTc and ICG-sulfo-OSu for PCSNs was verified by TLC before each experiment (D). Two-microliter spots of the grafted PCSNs were visualized on TLC plates for NIR fluorescence imaging (above) and contact radiography (below). Verification of the complete graft was confirmed by the lack of flares on either plate.

Figure 3

Lymphoscintigraphy of the rats and lymph node dissection in the neck. Lymphoscintigraphy of the rats (A, left). The nanoparticle solution was injected into the tongue and the left upper limb. Rat B was administered 7.4 MBq of PCSN probes in the tongue and the left upper limb; rat A was injected with a smaller volume (3.7 MBq) in the tongue. The larger injection volume (rat B) revealed an increased uptake in the neck (arrow), with a marked increase in uptake at the site of injection in rat B. A small semiconductor gamma camera clearly displayed the uptake in the neck with an acquisition time of 30 s (A, right). Lymph nodes were dissected from the neck of rat B (B, left). Distinct ICG fluorescent lymph nodes in the neck were observed in real time with anatomical resolution (B, right).

Figure 4

Lymph nodes from the dissected neck and serial scintigraphy. Four lymph nodes are shown from the dissected neck of a rat (A, upper left). Only one lymph node was observed as fluorescent in the dissected neck (A, upper right). Four lymph nodes were excised (A, lower left). Fine spatial distribution of the fluorescence was observed in one of the four lymph nodes (A, lower right). Serial scintigraphy of a rat after PCSN probe injection. Increased neck accumulation was observed at 10, 20, 30, 40, 50, and 60 min after injection; these time points are numbered 1 to 6, respectively (B).

Figure 5

Excised lymph nodes from rats B and D and contact radiography. Two excised lymph nodes from rat B are shown (A, left). Both lymph nodes were fluorescent (A, center). Contact radiography indicated that the nodes showing increased radioactivity also demonstrated increased levels of fluorescence (A, right). Four excised lymph nodes of rat D are shown (B, left). Two of the four lymph nodes were fluorescent. Fine spatial distribution of the fluorescence was observed in the lymph nodes (B, center). Contact radiography indicated a high level of accumulation of ^99mTc in the fluorescent lymph nodes (B, right).

Figure 6

Fifteen excised lymph nodes were divided into two groups based on their fluorescence intensity. The radioactivity of the excised lymph nodes relative to the initial injected radioactivity was compared between the high-fluorescence-intensity group (four lymph nodes) and the no-fluorescence-intensity group (11 lymph nodes). The high-fluorescence group also exhibited a high percentage of radioactivity (p < 0.05).

Figure 7

Toluidine blue staining, TEM of a macrophage, and Energy-dispersive spectroscopy. Toluidine blue staining shows a blood vessel and macrophages in a lymph node (A). TEM of a macrophage (C, D). High-power images revealed small, black granules in the macrophages. The granules of approximately 10 nm in diameter concentrated in the phagosomes with a neighboring mitochondria that did not contain granules inside (shown at the lower left corner of the panel C). There were also very few granules in the surrounding cellular components (C). Energy-dispersive spectroscopy of TEM. The arrow shows a Si (silicate) peak (B).