Development of a Hybrid Nanoprobe for Triple-Modality MR/SPECT/Optical Fluorescence Imaging

Abstract

Hybrid clinical imaging is an emerging technology, which improves disease diagnosis by combining already existing technologies. With the combination of high-resolution morphological imaging, i.e., MRI/CT, and high-sensitive molecular detection offered by SPECT/PET/Optical, physicians can detect disease progression at an early stage and design patient-specific treatments. To fully exploit the possibilities of hybrid imaging a hybrid probe compatible with each imaging technology is required. Here, we present a hybrid nanoprobe for triple modality MR/SPECT/Fluorescence imaging. Our imaging agent is comprised of superparamagnetic iron oxide nanoparticles (SPIONs), labeled with (99m)Tc and an Alexa fluorophore (AF), together forming (99m)Tc-AF-SPIONs. The agent was stable in human serum, and, after subcutaneous injection in the hind paw of Wistar rats, showed to be highly specific by accumulating in the sentinel lymph node. All three modalities clearly visualized the imaging agent. Our results show that a single imaging agent can be used for hybrid imaging. The use of a single hybrid contrast agent permits simultaneous hybrid imaging and, more conventionally, allow for single modality imaging at different time points. For example, a hybrid contrast agent enables pre-operative planning, intra-operative guidance, and post-operative evaluation with the same contrast agent.

Keywords: MR; SLN; SPECT; SPION; fluorescence imaging; magnetic resonance imaging; optical.

Figure 1

Transmission electron microscopy images of nanoparticles. (a) SPIONs in 0.9% saline buffer, pH 7 and (b) ^99mTc-AF-SPIONs after incubation in human serum at room temperature. The images demonstrate that the size of the nanoparticles is not affected by the labeling procedure including both radionuclides and fluorescent dye. No aggregations of the particles have been observed after incubation of ^99mTc-AF-SPIONs in human serum.

Figure 2

Representative coronal MR images of white Wistar rat injected subcutaneously with ^99mTc-AF-SPIONs in the right hind paw. Accumulation of the ^99mTc-AF-SPIONs in SLN can clearly visualized using (a) SE and (b) GRE sequences (white arrows).

Figure 3

SPECT/CT image of the same animal shown by MR in Figure 2. The arrows depict the injection site and accumulation of the ^99mTc-AF-SPIONs in SLN. SPECT is less affected by attenuation compared with optical imaging, therefore, is an invaluable tool to quantify and study the biodistribution of the newly developed agent.

Figure 4

Optical fluorescence image visualizing the SLN. High signal to background ratio is demonstrated which encourage for possible translation of this approach to clinical applications.

Figure 5

Intraoperative identification of the SLN using ^99mTc-AF-SPIONs. (a) Similar to the clinical procedure using blue dye, ^99mTc-AF-SPIONs stain the SLN green, which makes it easy to be identified during surgery. (b) The reference node from the collateral side of the animal. (c) The resected SLN.

Figure 6

Biodistribution of ^99mTc-AF-SPIONs in two white Wistar rats, 5 h post injection.

Figure 7

Microscopy images of a cryosectioned SLN. (a) Coronal section (20 µm) of a half SLN indicating the microdistribution of the nanoparticles within the SLN. The blue structures are cell nucleus and the red light is emitted from the ^99mTc-AF-SPIONs. The nanoparticles accumulate in the cortex and within the medullary sinuses. (b) Image visualizing the cortex of the SLN and indicating that the nanoparticles are mostly located extracellularly in comparison with the medullary sinus where the nanoparticles seem to be located within the macrophages. (c) and (d) bright field images of the SLN corresponding to (a) respectively (b) which show the orientation and anatomy.