3,7-bis(dimethylamino)-phenothiazin-5-ium chloride

A sentinel lymph node (SLN)is the first lymph node showing presence of metastasized cancer cells from a tumor. An SLN biopsy (SLNB) is the method of choice to stage breast cancer (i.e., to describe the stage of cancer progress using numbers I to IV, going from low to highest progression) in patients exhibiting clinically negative (i.e., without metastases) axillary lymph nodes (1, 2). Once detected and confirmed, the metastatic spread of the cancer from the primary tumor to the axillary lymph nodes through the lymphatic system is then tracked with either a radiolabeled sulfur colloid and/or a blue dye (isosulfan blue or methylene blue; neither of these dyes are approved by the United States Food and Drug Administration (FDA) for this application) (2). After visual confirmation, the SLN is surgically resected, and the definitive presence of cancer is established with histological methods. Although the SLNB procedure is minimally invasive, it has a 5%–10% false negative rate of detecting cancer and can possibly lead to lymphedema, seroma formation (collection of fluid under the skin), injury to local sensory nerves, and a reduction in the patient's range of motion (1, 2). In some cases, ultrasonography (US), which is the detection of high frequency sound waves, is used to identify a SLN, and a biopsy is obtained with a fine-needle aspiration biopsy (FNAB). The biopsy sample is then subjected to molecular analysis using the real-time reverse transcription-polymerase chain reaction to detect cancer in the tissue from the breast. A limitation of using US to identify a SLN is that it can only determine the size and shape of the lymph node but cannot specifically distinguish between a cancerous and a non-cancerous lymph node; this limitation reduces the sensitivity of the FNAB procedure to detect a malignancy (3). Photoacoustic imaging (PAI) is a noninvasive method that does not involve the use of ionizing radiation for the detection of cancer. This technique, which uses a combination of the optical and US imaging modalities to detect malignant lesions, angiogenesis, etc (4), has been described in detail elsewhere (5). In brief, application of a pulse of light to biological tissue results in the generation of ultrasonic waves (photoacoustic waves) due to the thermoelastic expansion and contraction of the tissue (i.e., a fraction of the light is converted into heat within the tissue). Subsequently, the photoacoustic waves are captured with a suitable device (transducer) that captures the US waves and converts them into an image. If used by itself, optical imaging has a limited imaging depth and spatial resolution because light is scattered by the tissue. As a result, images generated with US alone have a low contrast and are speckled (5). The main advantage of using PAI is that the depth and resolution of an image can be improved by controlling the detection frequency of the transducer, and the quality of the images can be further enhanced by using intrinsic contrast agents (e.g., melanin and hemoglobin) or exogenous contrast agents (e.g., an organic dye such as methylene blue, nanoparticles, etc.) (5). Methylene blue is an aromatic compound that has many applications in biology and medicine. In the laboratory, methylene blue is often used to stain blood cells for histological investigations, to detect nucleic acids on blots, and to determine the viability of yeast cells. Although methylene blue is not approved for clinical use by the FDA, it is available commercially in the United States for off label use as an intravenous injection to treat methemoglobinemia. Methylene blue is also under investigation in clinical trials to treat osteoarthritis, to detect invasive aspergillosis, and to map and localize SLN in breast and thyroid cancer patients. In several European countries, a procedure using methylene blue has been approved to reduce the pathogen load (e.g., viruses such as HIV and hepatitis B and C) in individual units of human plasma to be used for transfusion purposes (6). It has been shown that methylene blue radiolabeled with astatine-211 is suitable for the diagnosis and selective treatment of melanoma in mice (7). Investigators have also shown that methylene blue encapsulated in nanoparticles can be used for imaging and photodynamic therapy in mice (exposure of methylene blue nanoparticles to high-energy light pulses results in the production of nascent oxygen that damages the nucleic acids in the cells) (8). Recently, in an effort to develop an alternative method to SLNB and FNAB for the detection of SLNs in breast cancer patients, methylene blue has been successfully used as a contrast agent to visualize SLN in rats with PAI (1, 2).