Dumych Tetiana, Lutsyk Maxym, Banski Mateusz, Yashchenko Antonina, Sojka Bartlomiej, Horbay Rostyslav, Lutsyk Alexander, Stoika Rostyslav, Misiewicz Jan, Podhorodecki Artur, Bilyy Rostyslav
Rostyslav Bilyy, Drahomnanov Str. 14/16, 79005, Lviv, Ukraine,
Croat Med J. 2014 Jun 1;55(3):186-94. doi: 10.3325/cmj.2014.55.186.
To develop specific fluorescent markers for melanoma tumor visualization, which would provide high selectivity and reversible binding pattern, by the use of carbohydrate-recognizing proteins, lectins, combined with the physical ability for imaging deep in the living tissues by utilizing red and near infrared fluorescent properties of specific rare-earth doped nanocrystals (NC).
B10F16 melanoma cells were inoculated to C57BL/6 mice for inducing experimental melanoma tumor. Tumors were removed and analyzed by lectin-histochemistry using LABA, PFA, PNA, HPA, SNA, GNA, and NPL lectins and stained with hematoxylin and eosin. NPL lectin was conjugated to fluorescent NaGdF4:Eu(3+)-COOH nanoparticles (5 nm) via zero length cross-linking reaction, and the conjugates were purified from unbound substances and then used for further visualization of histological samples. Fluorescent microscopy was used to visualize NPL-NaGdF4:Eu(3+) with the fluorescent emission at 600-720 nm range.
NPL lectin selectively recognized regions of undifferentiated melanoblasts surrounding neoangiogenic foci inside melanoma tumor, PNA lectin recognized differentiated melanoblasts, and LCA and WGA were bound to tumor stroma regions. NPL-NaGdF4:Eu(3+) conjugated NC were efficiently detecting newly formed regions of melanoma tumor, confirmed by fluorescent microscopy in visible and near infrared mode. These conjugates possessed high photostability and were compatible with convenient xylene-based mounting systems and preserved intensive fluorescent signal at samples storage for at least 6 months.
NPL lectin-NaGdF4:Eu(3+) conjugated NC permitted distinct identification of contours of the melanoma tissue on histological sections using red excitation at 590-610 nm and near infrared emission of 700-720 nm. These data are of potential practical significance for development of glycans-conjugated nanoparticles to be used for in vivo visualization of melanoma tumor.
通过使用碳水化合物识别蛋白（凝集素），结合利用特定稀土掺杂纳米晶体（NC）的红色和近红外荧光特性在活组织深部成像的物理能力，开发用于黑色素瘤肿瘤可视化的特异性荧光标记物，该标记物应具有高选择性和可逆结合模式。
将B10F16黑色素瘤细胞接种到C57BL/6小鼠体内以诱导实验性黑色素瘤肿瘤。切除肿瘤并使用LABA、PFA、PNA、HPA、SNA、GNA和NPL凝集素通过凝集素组织化学进行分析，并用苏木精和伊红染色。通过零长度交联反应将NPL凝集素与荧光NaGdF4:Eu(3+)-COOH纳米颗粒（5纳米）偶联，从未结合的物质中纯化偶联物，然后用于组织学样本的进一步可视化。使用荧光显微镜在600 - 720纳米范围内的荧光发射来可视化NPL-NaGdF4:Eu(3+)。
NPL凝集素选择性识别黑色素瘤肿瘤内新血管生成灶周围未分化黑素母细胞区域，PNA凝集素识别分化的黑素母细胞，LCA和WGA与肿瘤基质区域结合。通过可见和近红外模式的荧光显微镜证实，NPL-NaGdF4:Eu(3+)偶联的NC能够有效检测黑色素瘤肿瘤新形成的区域。这些偶联物具有高光稳定性，与方便的基于二甲苯的封片系统兼容，并且在样本储存至少6个月时仍保留强烈的荧光信号。
NPL凝集素-NaGdF4:Eu(3+)偶联的NC允许在组织学切片上使用590 - 610纳米的红色激发光和700 - 720纳米的近红外发射光清晰识别黑色素瘤组织的轮廓。这些数据对于开发用于黑色素瘤肿瘤体内可视化的聚糖偶联纳米颗粒具有潜在的实际意义。
