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Tyrosinase (TYR) is the key enzyme regulating melanin production in hair and skin in humans. Although melanin displays maximal optical absorption at ultraviolet wavelengths, melanin has a broad spectrum of optical absorption with significant absorption at some near infrared wavelengths that have strong tissue penetrating properties. TYR has prior been shown to be a reporter gene. In addition, the recent study tests show that TYR is introduced into cells through gene transfer; and then the expressed tyrosinase catalyzes the synthesis of melanin from tyrosine precursors. The resulting gene product, melanin, can be imaged by three modalities including magnetic resonance imaging (MRI), photoacoustic imaging (PAI)and positron emission tomography (PET) (Figure 1).
Figure 1. Multimodality molecular imaging of the tyrosinase reporter gene system.
MRI of tyrosinase reporter gene
Because of a number of channels within melanin granules, melanin has a high affinity for many metal ions including Fe3+, Ca2+ and Mg2+ and can thus serve as a transporter of metal ions. In fact, pigmented tissues usually contain a large amount of metal ions in vivo. Clinical observations have shown that melanotic melanomas are connected with hyperintensity on T1-weighted MRI images. Therefore, TYR was previously proposed to be a reporter gene for MRI due to the good binding capability of melanin with Fe3+. The bound metal ions create a substantial increase in T1 signals and can be exploited as a contrast mechanism in MRI. Previous studies of using melanin as a MRI contrast agent were only carried out in cells. However, in the recent study, they successfully demonstrated that TYR can be used as a MRI reporter both in vivo and in vitro while achieving excellent tumor contrast.
PAI of tyrosinase reporter gene
PAI is an emerging hybrid molecular imaging modality capable of producing in vivo 3D images with higher spatial resolution than existing optical imaging modalities. The physical mechanism behind this non-invasive imaging modality is the photoacoustic effect. PAI can also provide functional and molecular information of diseases in real time by using endogenous or exogenous photoacoustic contrast agents. Thus, it is highly desirable to develop new imaging probes for PAI. Melanin has a broad optical absorption spectrum with significant absorption at near-infrared (NIR) wavelengths, which allows for good tissue penetration. Due to this unique property, melanin has been demonstrated to be an excellent endogenous contrast agent for PAI. Currently, the use of TYR as a reporter gene for PAI was explored, since TYR is the modulator of the synthesis of melanin. Nevertheless, in the prior studies, researchers only used TYR as a single modality reporter (PAI) and the potential of melanin as a multimodality reporter was not investigated. Recently, researchers systematically studied the sensitivity of PAI and carried out in vivo imaging in tumor mice models. High sensitivity and cell quantification capability using PAI was achieved in this study. In practical terms, the sensitivity was high enough to detect a concentration of MCF-7-TYR cells, which indicates that TYR reporter strategy can be used to detect a very small number of cells using PAI.
PET of tyrosinase reporter gene
Nuclear imaging modalities such as PET and SPECT have very high sensitivity and no limitation of tissue penetration. Recently, benzamide analogs have been shown to be the most promising melanin targeted imaging agents. Particularly, F-P3BZA appears to be a favorable agent due to its easy one-step radiochemical synthesis and excellent performance in vivo. Therefore, this probe can be effectively used to detect TYR reporter gene expression via melanin production, which is also supported by results of the cell uptake experiments and in vivo small animal imaging studies.
Figure 2. In vivo multimodality imaging of tumor bearing mice with PAI (B), MRI (C) and PET (D).
Moreover, melanin is a pigment and the expression of TYR reporter can be visually assessed by the color of cell pellets. Therefore, colorimetric assay can also be used for measurement of TYR reporter expression. This characteristic adds to the simplicity and ease-of-use of the TYR reporter strategy. In addition to serving as a reporter gene, TYR can also be used as a potential therapeutic gene. Recently, benzamide compounds and melanin-binding peptides labeled with therapeutic radionuclides have been actively pursued for melanin targeted melanoma treatment. Thus, the melanin induced by TYR gene in cancer cells can be regarded as an attractive target for some of these promising agents.