Turn-on Rhodamine Glycoconjugates Enable Real-Time GLUT Activity Monitoring in Live Cells and In Vivo.

The direct relationship between facilitative glucose transporters (GLUTs) and metabolic diseases opens new avenues for sensing metabolic deregulations and drives the development of molecular probes for GLUT-targeted detection of metabolic diseases. Radiotracer-based molecular imaging probes have been effectively utilized in reporting alterations in sugar uptake as an indication of metabolic deregulations, cancer development, or inflammation. Progress in developing fluorophore-based tools facilitated GLUT-specific analyses using more accessible fluorescence-based instrumentation. However, restrictions on the emission range of fluorophores and the requirement for substantial post-treatments to reduce background fluorescence have brought to light the critical directions for improvement of the technology for broader use in screening applications. Here we present turn-on GLUT activity reporters activated upon cells' internalization. We demonstrate a specific delivery of a sizable rhodamine B fluorophore through GLUT5 and showcase a stringent requirement in conjugate structure for maintaining a GLUT-specific uptake. With the turn-on GLUT probes, we demonstrate the feasibility of high-throughput fluorescence microscopy and flow cytometry-based GLUT activity screening in live cells and the probes' applicability for assessing sugar uptake alterations in vivo.

Highly Sensitive Cyanine Dyes for Rapid Sensing of NAD(P)H in Mitochondria and First-Instar Larvae of Drosophila melanogaster.

We have developed two highly sensitive cyanine dyes, which we refer to as probes A and B. These dyes are capable of quick and sensitive sensing of NAD(P)H. The dyes were fabricated by connecting benzothiazolium and 2,3-dimethylnaphtho[1,2-d]thiazol-3-ium units to 3-quinolinium through a vinyl bond. In the absence of NAD(P)H, both probes have low fluorescence and absorption peaks at 370 and 400 nm, correspondingly. This is because of their two electron-withdrawing acceptor systems with high charge densities. However, when NAD(P)H reduces the probes' electron-withdrawing 3-quinolinium units to electron-donating 1,4-dihydroquinoline units, the probes absorb at 533 and 535 nm and fluoresce at 572 and 586 nm for A and B correspondingly. This creates well-defined donor-π-acceptor cyanine dyes. We successfully used probe A to monitor NAD(P)H levels in live cells during glycolysis, under hypoxic conditions induced by CoCl2 treatment and after treatment with cancer drugs, including cisplatin, camptothecin, and gemcitabine. Probe A was also employed to visualize NAD(P)H in Drosophila melanogaster first-instar larvae. We observed an increase in NAD(P)H levels in A549 cancer cells both under hypoxic conditions and after treatment with cancer drugs, including cisplatin, camptothecin, and gemcitabine.

Near-infrared rhodol dyes bearing salicylaldehyde moieties for ratiometric pH sensing in live cells during mitophagy and under hypoxia conditions.

We describe a simple but efficient approach to make fluorescent probes A and B based on rhodol dyes incorporated with salicyaldehyde moiety for monitoring pH changes in mitochondria under oxidative stresses and hypoxia conditions, and for tracking mitophagy processes. Probes A and B possess pKa values (pKa ≈ 6.41 and 6.83 respectively) near physiological pH and exhibit decent mitochondria-targeted capabilities, low cytotoxicity, and useful ratiometric and reversible pH responses, which make the probes appropriate for monitoring pH fluctuations of mitochondria in living cells with built-in calibration feature for quantitative analysis. The probes have been effectively useful for the ratiometric determination of pH variations of mitochondria under the stimuli of carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP), hydrogen peroxide (H2O2), and N-acetyl cysteine (NAC), and during mitophagy triggered by cell nutrient deprivation, and under hypoxia conditions with cobalt chloride (CoCl2) treatment in living cells. In addition, probe A was efficient in visualizing pH changes in the larvae of fruit flies.