Near-infrared Absorption and Emission Probes with Optimal Connection Bridges for Live Monitoring of NAD(P)H Dynamics in Living Systems.

Two NAD(P)H-biosensing probes consisting of 1,3,3-trimethyl-3H-indolium and 3-quinolinium acceptors, linked by thiophene, A, and 3,4-ethylenedioxythiophene, B, bridges are detailed. We synthesized probes C and D, replacing the thiophene connection in probe A with phenyl and 2,1,3-benzothiadiazole units, respectively. Probe E was prepared by substituting probe A's 3-quinolinium unit with a 1-methylquinoxalin-1-ium unit. Solutions are non-fluorescent but in the presence of NADH, exhibit near-infrared fluorescence at 742.1 nm and 727.2 nm for probes A and B, respectively, and generate absorbance signals at 690.6 nm and 685.9 nm. In contrast, probes C and D displayed pronounced interference from NADH fluorescence at 450 nm, whereas probe E exhibited minimal fluorescence alterations in response to NAD(P)H. Pre-treatment of A549 cells with glucose in the presence of probe A led to a significant increase in fluorescence intensity. Additionally, subjecting probe A to lactate and pyruvate molecules resulted in opposite changes in NAD(P)H levels, with lactate causing a substantial increase in fluorescence intensity, conversely, pyruvate resulted in a sharp decrease. Treatment of A549 cells with varying concentrations of the drugs cisplatin, gemcitabine, and camptothecin (5, 10, and 20 µM) led to a concentration-dependent increase in intracellular fluorescence intensity, signifying a rise in NAD(P)H levels. Finally, fruit fly larvae were treated with different concentrations of NADH and cisplatin illustrating applicability to live organisms. The results demonstrated a direct correlation between fluorescence intensity and the concentration of NADH and cisplatin, respectively, further confirming the efficacy of probe A in sensing changes in NAD(P)H levels within a whole organism.

Sensitive monitoring of NAD(P)H levels within cancer cells using mitochondria-targeted near-infrared cyanine dyes with optimized electron-withdrawing acceptors.

A series of near-infrared fluorescent probes, labeled A to E, were developed by combining electron-rich thiophene and 3,4-ethylenedioxythiophene bridges with 3-quinolinium and various electron deficient groups, enabling the sensing of NAD(P)H. Probes A and B exhibit absorptions and emissions in the near-infrared range, offering advantages such as minimal interference from autofluorescence, negligible photo impairment in cells and tissues, and exceptional tissue penetration. These probes show negligible fluorescence when NADH is not present, and their absorption maxima are at 438 nm and 470 nm, respectively. In contrast, probes C-E feature absorption maxima at 450, 334 and 581 nm, respectively. Added NADH triggers the transformation of the electron-deficient 3-quinolinium units into electron-rich 1,4-dihydroquinoline units resulting in fluorescence responses which were established at 748, 730, 575, 625 and 661 for probes AH-EH, respectively, at detection limits of 0.15 µM and 0.07 µM for probes A and B, respectively. Optimized geometries based on theoretical calculations reveal non-planar geometries for probes A-E due to twisting of the 3-quinolinium and benzothiazolium units bonded to the central thiophene group, which all attain planarity upon addition of hydride resulting in absorption and fluorescence in the near-IR region for probes AH and BH in contrast to probes CH-EH which depict fluorescence in the visible range. Probe A has been successfully employed to monitor NAD(P)H levels in glycolysis and specific mitochondrial targeting. Furthermore, it has been used to assess the influence of lactate and pyruvate on the levels of NAD(P)H, to explore how hypoxia in cancer cells can elevate levels of NAD(P)H, and to visualize changes in levels of NAD(P)H under hypoxic conditions with CoCl2 treatment. Additionally, probe A has facilitated the examination of the potential impact of chemotherapy drugs, namely gemcitabine, camptothecin, and cisplatin, on metabolic processes and energy generation within cancer cells by affecting NAD(P)H levels. Treatment of A549 cancer cells with these drugs has been shown to increase NAD(P)H levels, which may contribute to their anticancer effects ultimately leading to programmed cell death or apoptosis. Moreover, probe A has been successfully employed in monitoring NAD(P)H level changes in D. melanogaster larvae treated with cisplatin.