Initial attempts to prepare iodo-methylene blue using the standard iodogen method resulted in the appearance of a major impurity that was not further characterized. To overcome this problem, we utilized KI/KIO3
under acidic conditions instead of the iodogen method 15
. Purification of the mixture yielded 125
I –MB 17
(), which has a retention time of 4.86 min and 97% purity based on the peak area. The unlabeled methylene blue eluted approximately 2 minutes before the iodinated species. A minor component that eluted at 4.95 min was the putative di-iodinated species that has been previously described 15
. Incorporation of 125
I in I-MB was quantitative and complete in 10 minutes. Because specific activity was not an issue in this study, the isolated 125
I-MB was not further purified.
FIGURE 1 Structures of methylene blue and iodo-methylene blue 17 (top). Reversed phase radio-chromatogram of 125I-iodo-methylene blue (bottom).
PAT images show clearly localized lymph nodes containing blue dye (). A control PAT image was acquired before injection of MB (). Soon after injection, MB accumulation in the SLN was detected photoacoustically. shows the PAT image of the MB-dyed SLN acquired at 10 min post-injection. An overlaid PAT and US image () provides both functional (MB uptake in the SLN) and structural information. Post-mortem photographs taken after PAT imaging confirmed MB uptake in the SLN (vide infra).
FIGURE 2 PAT imaging of lymph nodes after injection of MB. A) Control PAT image obtained before MB injection. B) PAT image taken at 10 minutes post-injection. C) Overlaid PAT (pseudo color) and US (gray scale) image. PAT, photoacoustic tomography; US, ultrasound (more ...)
SPECT imaging showed localized signal from 125I-MB in the axillary region within 1 h after injection (). The high intensity regions at the injection site, in the forelimb and at the location of the axillary lymph nodes were easily distinguished, allowing accurate mapping of the lymph fields. Approximately 9% (17.7+/−0.48 µCi, n=3) of the injected dose was detected in the axillary lymph node within 1 h after injection while almost no signal was detected in the region of the contralateral lymph node (). High uptake was confirmed by planar scintigraphy and post-mortem optical imaging (vide infra).
FIGURE 3 SPECT/CT projection images of rat acquired 1 h after subcutaneous injection of 125I-MB in the left forepaw. * indicates location of injection and crosshairs indicate location of signal from axillary lymph node. The red arrow indicates the contralateral (more ...)
We further validated that both PAT and SPECT signals originated from the same lymph node. Since our PAT and SPECT/CT imaging systems are not co-localized in the same lab area, we explored a multimodal imaging platform. In principle, 125I-MB can provide
photoacoustic, fluorescence, and gamma imaging signals. However, it was difficult to detect MB fluorescence through the skin by planar reflectance optical imaging. After removing the skin in the MB injection area, the lymph node and vessels were detected visually by dark blue stains (). The lymph tracts leading to the lymph node can be seen as well. Even with the exposed skin, only low fluorescence signal was detected in the forelimb and in the lymphatic vasculature, but was particularly dim from the area of the lymph node. This low nodal fluorescence could be attributed to the low fluorescence quantum yield of MB and likely quenching within the highly stained lymph nodes. The strength of the PAT signal suggests that much lower concentrations of MB can be used in future studies.
FIGURE 4 Post-mortem optical and planar scintigraphy of rat 2 h after subcutaneous injection of 125I-MB in the left forepaw. A) The MB can be clearly seen as blue coloring in the forepaw near the site of injection (*) and at the first lymph node after removal (more ...)
Unlike fluorescence imaging, planar gamma scintigraphy clearly showed high intensity from the left paw, lymphatic vessel, and lymph node () corresponding with the dark blue color observed in . The animal was euthanized, and then the dissected lymph nodes from the left axillary region were removed. After dissection, the first lymph node is distinctly blue from dye accumulation while the following nodes are free of the dye (). Thus, the post-mortem photograph taken after PAT imaging confirmed MB uptake in the SLN. Similarly, the ex-vivo planar scintigraphy confirmed that the first lymph node is the source of the SPECT signal, demonstrating that 125I-MB could serve as a reliable molecular imaging probe for in vivo validation of PAT in clinical trials.