Nanochemistry and biotechnology

NIR pentamethine cyanine dyes as fluorescent probes for live-cell imaging
Aristova D.1, Kot Y.2, Chernii S.1, Kot K.2, Kompaniiets A.2, Yarmoluk S.1
1Institute of Molecular Biology and Genetics NASU, 150 Zabolotnogo St., 03143 Kyiv, Ukraine.
E-mail: dar.arist@gmail.com
2V.N. Karazin Kharkiv National University, 4 Svobody Sq., 61022 Kharkiv, Ukraine.

Near-infrared (NIR; 650–900 nm) fluorescence imaging is an active and promising area for live-cell imaging. The NIR-emitted fluorescent dyes offer low energy of the excitation light, helping to avoid the phototoxicity effect and unwanted autofluorescent background. Therefore, it was decided as a promising study to evaluate NIR-emitted (675 – 710 nm) pentamethine cyanine dyes as fluorescent probes for live-cell visualization by fluorescence microscopy. 
Spectral-luminescent properties of the dyes in a free state and the presence of nucleic acids and serum albumins were characterized. Dyes possess low to moderate fluorescence intensity in a free state, but it increases while binding to serum albumins up to 160 times with a quantum yield value equal to 42%. We have studied the ability of the dyes to stain live mesenchymal stem cells from rat bone marrow (MSC) at a concentration 1µM. The dyes can penetrate the cell but have different selectivity to cell compartments. Dyes 1753Sl and 1759Sl accumulate in intracellular vesicles. Furthermore, we have confirmed that the dye 1753Sl visualizes autophagosomes and can be a marker of cell aging. At the same time, cyanine dyes 1756Sl and 1872Sl accumulate in cell mitochondria. The selectivity of 1872Sl towards mitochondria does not depend on the depolarization degree of the mitochondrial membrane, while 1756Sl stains only mitochondria with depolarized membrane.
Besides this, we performed a cytotoxicity study that included measuring the level of reactive oxygen species (ROS), determining the degree of damage to the plasma membrane of cells, and detecting apoptosis. The presence of cyanine dyes 1753SI, 1756SI, 1872SI, and 1759SI at a working concentration of 1µM does not damage the plasma membrane, does not increase ROS content, and does not induce apoptosis of bone marrow mesenchymal stromal cells. 
Thus, due to low cytotoxicity and selectivity to cell compartments, we can suggest the studied NIR-emitted pentamethine cyanine dyes as potential fluorescent probes to visualize live cells by fluorescence microscopy.