Preparation of a multi-modal agent for sentinel lymph node mapping using Evans blue and ^99mTc-labeled mannosylated...

The identification of sentinel lymph nodes (SLNs) is important in deciding the resection range during surgery. ^99mTc-labeled mannosylated human serum albumin ([^99mTc]Tc-MSA) is a radiopharmaceutical developed for SLN detection by targeting macrophages. Evans blue (EB) is a blue dye binding strongly to albumin and has been used for SLN detection. [^99mTc]Tc-MSA-EB conjugate was prepared as a multi-modal imaging agent and tested its performance by visual investigation, fluorescence imaging and SPECT/CT for SLN mapping in mice.

EB was mixed with various concentration of MSA to prepare MSA-EB conjugates. The binding efficiencies were determined using thin-layer chromatography. The UV-VIS spectra and molar extinction coefficient of the conjugate were obtained. The fluorescence was monitored at the excitation wavelength range 420-780 nm and the emission wavelength range 520-845 nm. The [^99mTc]Tc-MSA-EB conjugate and EB were injected into the footpads of normal BALB/c mice to check the lymph node (LN) uptakes. The visible, fluorescence, and SPECT/CT images were obtained after injection.

The conjugation of EB with MSA increased by time and was saturated within 10 min. The molar extinction coefficient of the conjugate was 99,259.3/M/cm at 620 nm. The uptake of conjugate into the popliteal LN after injection into the footpads of mice was investigated visually and fluorescence imaging. SPECT/CT images showed that the standardized uptake values of [^99mTc]Tc-MSA-EB conjugate in popliteal LN were about 4 times higher than in sciatic LN at all timepoints. It was confirmed by investigating resected LN that the blue color, fluorescence, and radioactivity of the [^99mTc]Tc-MSA-EB conjugate were retained only on the LN and did not spread to adjoining tissues.

[^99mTc]Tc-MSA-EB conjugate has a great potential as a multi-modal SLN mapping agent which could be detected by visual investigation, fluorescence imaging, and SPECT/CT.