Near-infrared fluorescent (NIRF) imaging of transferrin receptor in murine infection model

Abstract

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Objectives: To evaluate the feasibility of Transferrin-Cy5.5 as a near-infrared imaging agent for the detection of inflammation

Methods: Apo-Transferrin in 0.1 M NaHCO3 at pH 9.0 was mixed with one equivalents of Cy5.5-NHS in DMSO anhydrous. After stirring at room temperature for 2 h, the reaction mixture was passed through a gel filtration PD-10 column. The iron incorporation was performed by addition of 1.25 ul 10 mM iron (III) citrate buffer per mg transferrin. Mice were injected with E. coli (1*106/0.1 ml) in the left thigh muscle. After 24 h the infection/inflammation site was clearly visible in the mice. MR images were obtained. The imaging protocol included fat-suppressed, T2-weighted coronal images of the infected thigh for locating and delineating the infection site. Fluorescence images were then acquired with time after administration of Transferrin-Cy5.5 (0.5-0.8 mg transferrin/mouse). Inflammation images were obtained from 30 min to 180 min after injection of NIRF probe.

Results: Transferrin protein has one or more lysine residues which is accessible to labeling Cy5.5-NHS. The molar ratio of Cy5.5 to transferrin was estimated to be 0.53:1. The yield of transferrin was typically 83-87%. NIRF images showed the liver and kindeys faintly. Abnormally NIRF signal was clearly visualized in infected tissue. Through co-injection of Transferrin-Cy5.5 and free holo-transferrin, specific binding of Transferrin-Cy5.5 to transferrin receptor on cells gathering inflammation area was confirmed.

Conclusions: Inflammation of murine model was clearly visualized using Transferrin-Cy5.5. Transferrin-Cy5.5 seems to be useful optical imaging agent for detection of inflammation.