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Upon subcutaneous administration, the dye is quickly taken up by the lymphatic vessels to the draining nodes. Once the lymphatic mapping is shown by the blue color of EB dye, subsequent sentinel node biopsy becomes possible, which is critical for studies of solid tumor metastasis and of regional immune responses following immunization [ 18 , 74 ].

Due to this mechanism of action, Bobin et al. Harrell et al. Zheng et al. Similarly, Tervala et al. In addition, they analyzed the lymphatic vessel function by the way of quantifying the leakage of EB dye into the blood. Except the EB dye, several other dyes are useful for identification of sentinel lymph nodes and include Chicago sky blue, Patent blue, and Trypan blue [ 27 , 77 ].

It was previously reported that when scintigraphy and EB dye are used in tandem, the false-negative rate for sentinel node localization is decreased compared with using either agent alone [ 77 ]. On the whole, EB dye is a promising method for the assessment of malignancy and lymphedema in humans and subsequent sentinel node biopsy, as well as the evaluation of lymphatic vessel function.

Most tumors are known to exhibit highly enhanced vascular permeability, which is considered to facilitate tumor growth and perhaps metastasis [ 12 , 15 ]. Visual demarcation of tumor margins during operation will provide greater accuracy in tumor resection. The use of a visible small molecular dye to address this challenge was suggested by Ozawa et al.

EB dye is perfectly in accord with this demand, because EB dye may bind strongly to albumin, extravasates, and remains for a prolonged time in the extravascular space due to the enhanced permeability and retention EPR effect of tumors [ 79 ]. Lots of articles using EB dye to demarcate tumor margins were published [ 12 , 30 , 80 , 81 ]. Prabhu et al. Likewise, a study by Elsen et al. Thus, the method of EB instillations combined with white-light cystoscopy could be a useful tool for diagnosing bladder cancer in clinical settings in the future.

Interestingly, by developing a liposomally nanoencapsulated Evans blue dye nano-EB , Roller [ 12 ] demonstrated that clearer tumor margins were demarcated with nano-EB in an invasive tumor model compared with unencapsulated EB.

This indicated that nano-EB could be deposited specifically to tumor tissue, which is normally considered as the tumor-specific feature of the EB dye [ 12 , 82 ].

As we know, liposomal nanocarriers as drug carriers have been extensively investigated in nanomedicine; the special structure of liposomes can facilitate itself, evading the reticuloendothelial system and prolonging the circulation time in the bloodstream [ 83 , 84 ]. Much effort has been made to design and optimize liposomal nanocarriers. For instance, incorporation of targeting moieties to tumors onto the liposomal may help to reduce the accumulation in nontarget organs [ 84 ].

Therefore, nano-EB may be used to provide accurate visual cues for the surgeon to intraoperatively delineate the tumor margins via two mechanisms: either via tumor EPR effect or via tumor-receptor targeting strategy [ 12 ]. In general, EB dye, especially liposomal nanocarriers-encapsulated Evans blue, could be a useful agent to localize and demarcate the solid tumors more precisely due to its selective accumulation in the tumor site.

This so-called tumor-specific feature may actually be attributed to the EPR effect that may be used to explain the mechanism of EB applications in many clinical settings. As we know, life and death are two forms of cellular existence. Cell death is an essential form in cell development.

Cell death includes at least two independent modes, that is, apoptosis and necrosis. In their study, EB dye leaked through damaged membranes and stained the dead cells but was excluded by viable cells. EB dye has been further investigated and used extensively for specifying cell death in microscopic studies [ 86 ]. Although EB dye has been shown to be reliable for the assessment of viability in plant cells, it remains unclear whether EB dye can be also used in mammalian cells.

In , Matsuda et al. In contrast, a study by Straub et al. Similarly, Miller et al. Klyen et al. Many researchers used EB dye to discriminate among injured cells in various animal models, such as the zebrafish models of neuromuscular disease [ 14 ], mouse models of muscular dystrophy [ 90 ] and experimental injury and repair [ 91 ], rat models of stroke and cardiomyocyte injury [ 92 ], myocardial infarction core in a rabbit model [ 32 ] Figure 1 , and reperfused partial liver infarction model in rats [ 33 ] Figure 2.

These findings implied the nature of necrosis-avidity for EB dye. Although the EB dye was considered as a necrosis-avid agent, the specific mechanism is still not clear [ 94 ]. Therefore, -EB could be a promising agent in the clinical setting to guide sentinel node biopsy in various solid tumors in the near future.

Taking full advantage of the albumin-binding feature of EB, Liu et al. These EB derivatives may be conjugated onto a therapeutic small molecule, peptide, or an oligonucleotide aptamer. Thus, they may be used as theranostics with a broad potential due to their improved half-life in the blood and reduced release.

These radiolabeled NEB derivatives were initially used as blood pool agents to evaluate myocardial infarction and vessel leakage in inflammation and tumors [ 96 ]. After local injection, 18 F-AlF-NEB can be used to visualize and detect the sentinel lymph nodes with a high signal-to-noise ratio. A preliminary study showed the potential of 68 Ga-NEB in differentiating hepatic hemangioma from other focal hepatic lesions [ 97 ]. When EB-maleimide, one of the truncated EB derivatives, was conjugated to antidiabetic drug exendin-4 denoted as Abextide , it showed great potential to treat type 2 diabetic mice due to the significantly extended biological half-life of Abextide through complexation with albumin in situ [ 25 , 26 ].

This strategy provides possibilities for the development of long-acting therapeutic drugs for other small molecules and biologics [ 26 ]. EB dye has been widely used for different applications in the clinic and research, but few researchers have considered the probable toxicity of the dye.

In most cases, few reports of adverse reactions were seen. However, it was reported by Gibson and Gregersen in that pulmonary embolism was observed after intravenous injection of EB in rats. In , Giger et al. In addition, Jackiewicz et al. Recently, an ex vivo experiment by Giansanti et al. They found damage of ARPE cells and RGC5 cells with halogen and xenon light exposure should be a concern when the dye is used during vitrectomy [ ]. The point that many researchers have easily ignored is that azo dyes, as a class, can potentially induce mutagenicity and carcinogenicity when split off or degraded into component aromatic amines, especially in the fetus.

Although there is a lack of reports about the carcinogenicity of EB, it is necessary to consider the possibility when using EB in clinical practice. In summary, the once widely used EB dye dilution method has gradually been replaced by newer methods, such as radiolabeled albumin and radiolabeled red cells, for the estimation of blood volume and cardiac output. The blood pool effect of EB dye is limited when evaluating perfused or hypoperfused myocardium, but its utility in assessing vascular permeability and BBB integrity has increased.

In addition, the use of radiolabeled EB and its derivatives will play an important role in clinical imaging of tumor lesions, evaluation of lymphatic disorders, and development of long-acting therapeutics. Importantly, the newly discovered necrosis-avid affinity of EB will facilitate the identification of necrotic tissues including myocardial infraction, cerebral stroke, degenerating muscular diseases, and even amyloidosis [ ], as well as the assessment of therapeutic efficacy and prognosis of solid tumors.

Although the mechanisms are still unclear and further investigation is required, EB dye as a vital stain has greatly contributed to biomedical research and may continue to benefit the development of medical practice and patient care. Therefore, the main advantage of new applications of Evans blue lies in its great potential use in clinical practice as mentioned above. The authors declare that there are no conflicts of interest regarding the publication of this paper.

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Academic Editor: Yuebing Wang. Received 06 Nov Accepted 06 Mar Published 22 Apr Abstract Evans blue EB dye has owned a long history as a biological dye and diagnostic agent since its first staining application by Herbert McLean Evans in Introduction Dating back to the early 20th century, lots of blue dyes were synthesized like Methylene blue, Patent blue, Trypan blue, and so forth, and a comparison of main blue dyes was given in Table 1 [ 1 — 4 ].

Table 1. Figure 1. Evaluation of myocardial infarction core MI-core , area at risk AAR , and salvageable zone SZ in a rabbit with reperfused MI by in vivo and ex vivo imaging techniques and dynamic imaging quantification [ 32 ]. Figure 2. Postmortem analysis of necrotic and viable liver from rats with reperfused partial liver infarction that received iodinelabeled monoiodohypericin followed by the necrosis-avid dye, Evans blue [ 33 ].

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