OBJECTIVE

To prepare the magnetic near infrared fluorescent (NIRF) bifunctional molecular probe with human holo-transferrin (Tf) as a targeted ligand and detect human transferrin receptor (hTfR) actively.

METHODS

Molecular probe Tf-cy5.5-IO was prepared and purified by conjugating Tf, superparamagnetic iron oxide (IO) and near infrared fluorescent dye (cy5.5). The particle size and morphology was determined by transmission electron microscopy (TEM), zeta potential and particle sizing analyzer. Human serum albumin (HSA) was used for conjugating with cy5.5 and IO as control. hMSCs and HeLa (as a positive control) were divided into 4 groups: A non-labeled, B Tf-cy5.5-IO, C HSA-cy5.5-IO and D competition assay to confirm the targeted connection. The fluorescent signals from intracellular probe were detected with laser scanning confocal microscope (LSCM) and flow cytometry. Intracellular iron was detected with iron concentration assay and TEM. MRI and NIRF imaging of 2×10(5) cells were performed respectively. Enhancements of R2 value and average intensity (AI) were analyzed qualitatively.

RESULTS

The conjugation between IO, Tf and cy5.5 was confirmed with a molar ratio of 1: 2.89: 7.89. The hyperdense aqueous diameter of probe was 23.39 ± 2.42 nm. LSCM showed the fluorescence from Tf-cy5.5-IO and cy3-labeled monoclonal antibody against hTfR in cells and two markers were localized in intracellular compartments of similar appearance. After co-incubating with Tf-cy5.5-IO, the intracellular iron and average intensity were significantly higher than cells of other groups (P < 0.01). MRI and NIRF images showed that, after incubation, intracellular Tf-cy5.5-IO decreased the T2WI signal of human mesenchymal stem cells (hMSCs) and AI on NIRF image increased. Enhancements of R2 value and AI were higher in B group than those in other groups (P < 0.05).

CONCLUSION

Tf-cy5.5-IO probe can recognize and conjugate with hTfR specifically. And targeted imaging in vitro of hTfR expressed in hMSCs may be performed by MRI and NIRF multimodal imaging.